HTP genomic engineering platform for improving fungal strains

ABSTRACT

A HTP genomic engineering platform for improving filamentous fungal cells that is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols is provided. This integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterative pattern recognition. Methods for isolating clonal populations derived from individual fungal spores are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.16/600,062, filed Oct. 11, 2019, (issued as U.S. Pat. No. 10,954,511 onMar. 23, 2021), which is a Continuation of International PCT ApplicationNo. PCT/US2018/036360, filed Jun. 6, 2018, which claims the benefit ofpriority to U.S. Provisional Application No. 62/515,907, filed on Jun.6, 2017, each of which is hereby incorporated by reference in itsentirety for all purposes.

FIELD

The present disclosure is directed to automated fungal genomicengineering. The disclosed automated genomic engineering platformentails the genetic manipulation of filamentous fungi to generate fungalproduction strains as well as facilitate purification thereof. Theresultant fungal production strains are well-suited for growth insub-merged cultures, e.g., for the large-scale production of products ofinterest (e.g., antibiotics, metabolites, proteins, etc.) for commercialapplications.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ZYMR_008_05US_SubSeqList_ST25.txt. The text fileis 52,485 bytes, was created on Mar. 19, 2021, and is being submittedelectronically via EFS-Web.

BACKGROUND

Eukaryotic cells are preferred organisms for the production ofpolypeptides and secondary metabolites. In fact, filamentous fungi arecapable of expressing native and heterologous proteins to high levels,making them well-suited for the large-scale production of enzymes andother proteins for industrial, pharmaceutical, animal health and foodand beverage applications. However, use of filamentous fungi forlarge-scale production of products of interest often requires geneticmanipulation of said fungi as well as use of automated machinery andequipment and certain aspects of the filamentous fungal life cycle canmake genetic manipulation and handling difficult.

For example, DNA introduced into a fungus integrates randomly within agenome, resulting in mostly random integrated DNA fragments, which quiteoften can be integrated as multiple tandem repeats (see for exampleCasqueiro et al., 1999, J. Bacteriol. 181:1181-1188). This uncontrolled“at random multiple integration” of an expression cassette can be apotentially detrimental process, which can lead to unwanted modificationof the genome of the host.

Additionally, present transfection systems for filamentous fungi can bevery laborious (see for review Fincham, 1989, Microbiol. Rev.53:148-170) and relatively small scale in nature. This can involveprotoplast formation, viscous liquid handling (i.e. polyethylene glycolsolutions), one-by-one swirling of glass tubes and subsequent selectiveplating. Further, conditions for protoplasting can be difficult todetermine and yields can often be quite low. Moreover, the protoplastscan contain multiple nuclei such that introduction of a desired geneticmanipulation can lead to the formation of heterokaryotic protoplaststhat can be difficult to separate from homokaryotic protoplasts.

Further, typical filamentous fungal cells, including those derived fromprotoplasts, grow as long fibers called hyphae that can form densenetworks of hyphae called mycelium. These hyphae can contain multiplenuclei that can differ from one another in genotype. The hyphae candifferentiate and form asexual spores that can be easily dispersed inthe air. If the hyphae contain nuclei of different genotypes, the sporeswill also contain a mixture of nuclei. Due to this aspect of fungalgrowth, genetic manipulation inherently results in a mixed populationthat must be purified to homogeneity in order to assess any effect ofthe genetic changes made. Further, in an automated environment, thespores can cause contamination of equipment that could negatively impactthe ability to purify strains and may contaminate any other workperformed on the equipment.

To mitigate the aerial dispersal of spores, the filamentous fungi can begrown in submerged cultures. However, the mycelium formed by hyphalfilamentous fungi growth in submerged cultures can affect therheological properties of the broth. Generally, the higher the viscosityof the broth, the less uniform the distribution of oxygen and nutrients,and the more energy required to agitate the culture. In some cases, theviscosity of the broth due to hyphal filamentous fungal growth becomessufficiently high to significantly interfere with the dissolution ofoxygen and nutrients, thereby adversely affecting the growth of thefungi and ultimately the yield and productivity of any desired productof interest.

Thus, there is a great need in the art for new methods of engineeringfilamentous fungi, which do not suffer from the aforementioned drawbacksinherent with traditional strain building programs in fungi and greatlyaccelerate the process of discovering and consolidating beneficialmutations.

SUMMARY OF THE DISCLOSURE

The present disclosure provides a high-throughput (HTP) genomicengineering platform for coenocytic organisms such as, for examplefilamentous fungi that does not suffer from the myriad of problemsassociated with traditional microbial strain improvement programs. Whilethe methods provided herein are tested in filamentous fungi, it iscontemplated that said methods can be applied to and/or utilized inother coenocytic organisms. In one embodiment, the filamentous fungus isselected from Achlya, Acremonium, Aspergillus, Aureobasidium,Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus,Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia,Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea,Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora,Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor,Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces,Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma,Verticillium, Volvariella species or teleomorphs, or anamorphs, andsynonyms or taxonomic equivalents thereof. Further to this embodiment,the filamentous fungus useful for the methods and HTP genomicengineering platform is Aspergillus niger.

Further, the HTP platform taught herein is able to rehabilitatefilamentous fungal strains that have accumulated non-beneficialmutations through decades of random mutagenesis-based strain improvementprograms.

The disclosure also provides for unique genomic engineering toolsets andprocedures, which undergird the HTP platform's functionality in afilamentous fungal system. The filamentous fungus can be an Aspergillusspecies. The Aspergillus can be A. niger.

The disclosed HTP genomic engineering platform is computationally drivenand integrates molecular biology, automation, and advanced machinelearning protocols. This integrative platform utilizes a suite of HTPmolecular tool sets to create HTP genetic design libraries, which arederived from, inter alia, scientific insight and iterative patternrecognition.

The taught HTP genetic design libraries function as drivers of thegenomic engineering process, by providing libraries of particulargenomic alterations for testing in filamentous fungal The microbesengineered utilizing a particular library, or combination of libraries,are efficiently screened in a HTP manner for a resultant outcome, e.g.production of a product of interest. This process of utilizing the HTPgenetic design libraries to define particular genomic alterations fortesting in a microbe and then subsequently screening host microbialgenomes harboring the alterations is implemented in an efficient anditerative manner. In some aspects, the iterative cycle or “rounds” ofgenomic engineering campaigns can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or moreiterations/cycles/rounds.

Thus, in some aspects, the present disclosure teaches methods ofconducting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875,900, 925, 950, 975, 1000 or more “rounds” of HTP genetic engineering(e.g., rounds of SNP swap, PRO swap, Terminator (STOP) swap, orcombinations thereof) in an filamentous fungal host system.

In some embodiments, the present disclosure teaches a linear approach,in which each subsequent HTP genetic engineering round is based ongenetic variation identified in the previous round of geneticengineering. In other embodiments the present disclosure teaches anon-linear approach, in which each subsequent HTP genetic engineeringround is based on genetic variation identified in any previous round ofgenetic engineering, including previously conducted analysis, andseparate HTP genetic engineering branches.

The data from these iterative cycles enables large scale data analyticsand pattern recognition, which is utilized by the integrative platformto inform subsequent rounds of HTP genetic design libraryimplementation. Consequently, the HTP genetic design libraries utilizedin the taught platform are highly dynamic tools that benefit from largescale data pattern recognition algorithms and become more informativethrough each iterative round of microbial engineering. Such a system hasnever been developed for filamentous fungal and is desperately needed inthe art.

In some embodiments, the genetic design libraries of the presentdisclosure comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825,850, 875, 900, 925, 950, 975, 1000 or more individual genetic changes(e.g., at least X number of promoter:gene combinations in the PRO swaplibrary).

In some embodiments, the present disclosure teaches a high-throughput(HTP) method of genomic engineering to evolve an filamentous fungalstrain to acquire a desired phenotype, comprising: a) perturbing thegenomes of an initial plurality of filamentous fungal strains having thesame strain background, to thereby create an initial HTP genetic designfilamentous fungal strain library comprising individual strains withunique genetic variations; b) screening and selecting individual strainsof the initial HTP genetic design filamentous fungal strain library forthe desired phenotype; c) providing a subsequent plurality offilamentous fungal microbes that each comprise a unique combination ofgenetic variation, said genetic variation selected from the geneticvariation present in at least two individual filamentous fungal strainsscreened in the preceding step, to thereby create a subsequent HTPgenetic design filamentous fungal strain library; d) screening andselecting individual filamentous fungal strains of the subsequent HTPgenetic design filamentous fungal strain library for the desiredphenotype; e) repeating steps c)-d) one or more times, in a linear ornon-linear fashion, until an filamentous fungal strain has acquired thedesired phenotype, wherein each subsequent iteration creates a new HTPgenetic design filamentous fungal strain library comprising individualFilamentous fungal strains harboring unique genetic variations that area combination of genetic variation selected from amongst at least twoindividual filamentous fungal strains of a preceding HTP genetic designfilamentous fungal strain library.

In some embodiments, the present disclosure teaches that the initial HTPgenetic design filamentous fungi strain library is at least one selectedfrom the group consisting of a promoter swap microbial strain library,SNP swap microbial strain library, start/stop codon microbial strainlibrary, optimized sequence microbial strain library, a terminator swapmicrobial strain library, or any combination thereof.

In some embodiments, the present disclosure teaches methods of making asubsequent plurality of filamentous fungi strains that each comprise aunique combination of genetic variations, wherein each of the combinedgenetic variations is derived from the initial HTP genetic designfilamentous fungi strain library or the HTP genetic design filamentousfungi strain library of the preceding step.

In some embodiments, the combination of genetic variations in thesubsequent plurality of filamentous fungi strains will comprise a subsetof all the possible combinations of the genetic variations in theinitial HTP genetic design filamentous fungi strain library or the HTPgenetic design filamentous fungi strain library of the preceding step.

In some embodiments, the present disclosure teaches that the subsequentHTP genetic design filamentous fungi strain library is a fullcombinatorial strain library derived from the genetic variations in theinitial HTP genetic design filamentous fungi strain library or the HTPgenetic design filamentous fungi strain library of the preceding step.

For example, if the prior HTP genetic design filamentous fungi strainlibrary only had genetic variations A, B, C, and D, then a partialcombinatorial of said variations could include a subsequent HTP geneticdesign filamentous fungi strain library comprising three strains witheach comprising either the AB, AC, or AD unique combinations of geneticvariations (order in which the mutations are represented isunimportant). A full combinatorial filamentous fungi strain libraryderived from the genetic variations of the HTP genetic design library ofthe preceding step would include six microbes, each comprising eitherAB, AC, AD, BC, BD, or CD unique combinations of genetic variations.

In some embodiments, the methods of the present disclosure teachperturbing the genome of filamentous fungi utilizing at least one methodselected from the group consisting of: random mutagenesis, targetedsequence insertions, targeted sequence deletions, targeted sequencereplacements, or any combination thereof.

In some embodiments of the presently disclosed methods, the initialplurality of filamentous fungi unique genetic variations derived from anindustrial production filamentous fungi strain.

In some embodiments of the presently disclosed methods, the initialplurality of filamentous fungi comprise industrial productionfilamentous fungi strains denoted S₁Gen₁ and any number of subsequentmicrobial generations derived therefrom denoted S_(n)Gen_(n).

In some embodiments, the present disclosure teaches a method forgenerating a SNP swap filamentous fungi strain library, comprising thesteps of: a) providing a reference filamentous fungi strain and a secondfilamentous fungi strain, wherein the second filamentous fungi straincomprises a plurality of identified genetic variations selected fromsingle nucleotide polymorphisms, DNA insertions, and DNA deletions,which are not present in the reference strain; b) perturbing the genomeof either the reference strain, or the second strain, to thereby createan initial SNP swap filamentous fungi strain library comprising aplurality of individual filamentous fungi strains with unique geneticvariations found within each strain of said plurality of individualstrains, wherein each of said unique genetic variations corresponds to asingle genetic variation selected from the plurality of identifiedgenetic variations between the reference strain and the second strain.

In some embodiments of a SNP swap library, the genome of the referencefilamentous fungi strain is perturbed to add one or more of theidentified single nucleotide polymorphisms, DNA insertions, or DNAdeletions, which are found in the second filamentous fungi strain.

In some embodiments of a SNP swap library, the genome of the secondfilamentous fungi strain is perturbed to remove one or more of theidentified single nucleotide polymorphisms, DNA insertions, or DNAdeletions, which are not found in the reference filamentous fungistrain.

In some embodiments, the genetic variations of the SNP swap library willcomprise a subset of all the genetic variations identified between thereference filamentous fungi strain and the second filamentous fungistrain.

In some embodiments, the genetic variations of the SNP swap library willcomprise all of the identified genetic variations identified between thereference filamentous fungi strain and the second filamentous fungistrain.

In some embodiments, the present disclosure teaches a method forrehabilitating and improving the phenotypic performance of an industrialfilamentous fungi strain, comprising the steps of: a) providing aparental lineage filamentous fungi strain and an industrial filamentousfungi strain derived therefrom, wherein the industrial strain comprisesa plurality of identified genetic variations selected from singlenucleotide polymorphisms, DNA insertions, and DNA deletions, not presentin the parental lineage strain; b) perturbing the genome of either theparental lineage strain, or the industrial strain, to thereby create aninitial SNP swap filamentous fungi strain library comprising a pluralityof individual strains with unique genetic variations found within eachstrain of said plurality of individual strains, wherein each of saidunique genetic variations corresponds to a single genetic variationselected from the plurality of identified genetic variations between theparental lineage strain and the industrial strain; c) screening andselecting individual strains of the initial SNP swap filamentous fungistrain library for phenotype performance improvements over a referencefilamentous fungi strain, thereby identifying unique genetic variationsthat confer said filamentous fungi strains with phenotype performanceimprovements; d) providing a subsequent plurality of filamentous fungistrains that each comprise a unique combination of genetic variation,said genetic variation selected from the genetic variation present in atleast two individual strains screened in the preceding step, to therebycreate a subsequent SNP swap filamentous fungi strain library; e)screening and selecting individual strains of the subsequent SNP swapfilamentous fungi strain library for phenotype performance improvementsover the reference strain, thereby identifying unique combinations ofgenetic variation that confer said filamentous fungi strains withadditional phenotype performance improvements; and f) repeating stepsd)-e) one or more times, in a linear or non-linear fashion, until astrain exhibits a desired level of improved phenotype performancecompared to the phenotype performance of the industrial filamentousfungi strain, wherein each subsequent iteration creates a new SNP swapfilamentous fungi strain library comprising individual microbial strainsharboring unique genetic variations that are a combination of geneticvariation selected from amongst at least two individual microbialstrains of a preceding SNP swap filamentous fungi strain library.

In some embodiments, the present disclosure teaches methods forrehabilitating and improving the phenotypic performance of an industrialfilamentous fungi strain, wherein the genome of the parental lineagefilamentous fungi strain is perturbed to add one or more of theidentified single nucleotide polymorphisms, DNA insertions, or DNAdeletions, which are found in the industrial filamentous fungi strain.

In some embodiments, the present disclosure teaches methods forrehabilitating and improving the phenotypic performance of an industrialfilamentous fungi strain, wherein the genome of the industrialfilamentous fungi strain is perturbed to remove one or more of theidentified single nucleotide polymorphisms, DNA insertions, or DNAdeletions, which are not found in the parental lineage filamentous fungistrain.

In some embodiments, the present disclosure teaches a method forgenerating a promoter swap filamentous fungi strain library, said methodcomprising the steps of: a) providing a plurality of target genesendogenous to a base filamentous fungi strain, and a promoter ladder,wherein said promoter ladder comprises a plurality of promotersexhibiting different expression profiles in the base filamentous fungistrain; b) engineering the genome of the base filamentous fungi strain,to thereby create an initial promoter swap filamentous fungi strainlibrary comprising a plurality of individual filamentous fungi strainswith unique genetic variations found within each strain of saidplurality of individual strains, wherein each of said unique geneticvariations comprises one of the promoters from the promoter ladderoperably linked to one of the target genes endogenous to the basefilamentous fungi strain.

In some embodiments, the present disclosure teaches a promoter swapmethod of genomic engineering to evolve an filamentous fungi strain toacquire a desired phenotype, said method comprising the steps of: a)providing a plurality of target genes endogenous to a base filamentousfungi strain, and a promoter ladder, wherein said promoter laddercomprises a plurality of promoters exhibiting different expressionprofiles in the base filamentous fungi strain; b) engineering the genomeof the base filamentous fungi strain, to thereby create an initialpromoter swap filamentous fungi strain library comprising a plurality ofindividual filamentous fungi strains with unique genetic variationsfound within each strain of said plurality of individual strains,wherein each of said unique genetic variations comprises one of thepromoters from the promoter ladder operably linked to one of the targetgenes endogenous to the base filamentous fungi strain; c) screening andselecting individual strains of the initial promoter swap filamentousfungi strain library for the desired phenotype; d) providing asubsequent plurality of filamentous fungi strains that each comprise aunique combination of genetic variation, said genetic variation selectedfrom the genetic variation present in at least two individual strainsscreened in the preceding step, to thereby create a subsequent promoterswap filamentous fungi strain library; e) screening and selectingindividual strains of the subsequent promoter swap filamentous fungistrain library for the desired phenotype; f) repeating steps d)-e) oneor more times, in a linear or non-linear fashion, until a microbe hasacquired the desired phenotype, wherein each subsequent iterationcreates a new promoter swap filamentous fungi strain library comprisingindividual strains harboring unique genetic variations that are acombination of genetic variation selected from amongst at least twoindividual strains of a preceding promoter swap filamentous fungi strainlibrary.

In some embodiments, the present disclosure teaches a method forgenerating a terminator swap filamentous fungi strain library, saidmethod comprising the steps of: a) providing a plurality of target genesendogenous to a base filamentous fungi strain, and a terminator ladder,wherein said terminator ladder comprises a plurality of terminatorsexhibiting different expression profiles in the base filamentous fungistrain; b) engineering the genome of the base filamentous fungi strain,to thereby create an initial terminator swap filamentous fungi strainlibrary comprising a plurality of individual strains with unique geneticvariations found within each strain of said plurality of individualstrains, wherein each of said unique genetic variations comprises one ofthe target genes endogenous to the base filamentous fungi strainoperably linked to one or more of the terminators from the terminatorladder.

In some embodiments, the present disclosure teaches a terminator swapmethod of genomic engineering to evolve an filamentous fungi strain toacquire a desired phenotype, said method comprising the steps of; a)providing a plurality of target genes endogenous to a base filamentousfungi strain, and a terminator ladder, wherein said terminator laddercomprises a plurality of terminators exhibiting different expressionprofiles in the base filamentous fungi strain; b) engineering the genomeof the base filamentous fungi strain, to thereby create an initialterminator swap filamentous fungi strain library comprising a pluralityof individual filamentous fungi strains with unique genetic variationsfound within each strain of said plurality of individual strains,wherein each of said unique genetic variations comprises one of thetarget genes endogenous to the base filamentous fungi strain operablylinked to one or more of the terminators from the terminator ladder; c)screening and selecting individual microbial strains of the initialterminator swap filamentous fungi strain library for the desiredphenotype; d) providing a subsequent plurality of filamentous fungistrains that each comprise a unique combination of genetic variation,said genetic variation selected from the genetic variation present in atleast two individual strains screened in the preceding step, to therebycreate a subsequent terminator swap filamentous fungi strain library; e)screening and selecting individual strains of the subsequent terminatorswap filamentous fungi strain library for the desired phenotype; f)repeating steps d)-e) one or more times, in a linear or non-linearfashion, until a microbe has acquired the desired phenotype, whereineach subsequent iteration creates a new terminator swap filamentousfungi strain library comprising individual strains harboring uniquegenetic variations that are a combination of genetic variation selectedfrom amongst at least two individual strains of a preceding terminatorswap filamentous fungi strain library.

In some embodiments, the present disclosure teaches iterativelyimproving the design of candidate filamentous fungi strains by (a)accessing a predictive model populated with a training set comprising(1) inputs representing genetic changes to one or more backgroundfilamentous fungi strains and (2) corresponding performance measures;(b) applying test inputs to the predictive model that represent geneticchanges, the test inputs corresponding to candidate filamentous fungistrains incorporating those genetic changes; (c) predicting phenotypicperformance of the candidate filamentous fungi strains based at least inpart upon the predictive model; (d) selecting a first subset of thecandidate filamentous fungi strains based at least in part upon theirpredicted performance; (e) obtaining measured phenotypic performance ofthe first subset of the candidate filamentous fungi strains; (f)obtaining a selection of a second subset of the candidate filamentousfungi strains based at least in part upon their measured phenotypicperformance; (g) adding to the training set of the predictive model (1)inputs corresponding to the selected second subset of candidatefilamentous fungi strains, along with (2) corresponding measuredperformance of the selected second subset of candidate filamentous fungistrains; and (h) repeating (b)-(g) until measured phenotypic performanceof at least one candidate filamentous fungi strain satisfies aperformance metric. In some cases, during a first application of testinputs to the predictive model, the genetic changes represented by thetest inputs comprise genetic changes to the one or more backgroundfilamentous fungi strains; and during subsequent applications of testinputs, the genetic changes represented by the test inputs comprisegenetic changes to candidate filamentous fungi strains within apreviously selected second subset of candidate filamentous fungistrains.

In some embodiments, selection of the first subset may be based onepistatic effects. This may be achieved by: during a first selection ofthe first subset: determining degrees of dissimilarity betweenperformance measures of the one or more background filamentous fungistrains in response to application of a plurality of respective inputsrepresenting genetic changes to the one or more background filamentousfungi strains; and selecting for inclusion in the first subset at leasttwo candidate filamentous fungi strains based at least in part upon thedegrees of dissimilarity in the performance measures of the one or morebackground filamentous fungi strains in response to application ofgenetic changes incorporated into the at least two candidate filamentousfungi strains.

In some embodiments, the present disclosure teaches applying epistaticeffects in the iterative improvement of candidate filamentous fungistrains, the method comprising: obtaining data representing measuredperformance in response to corresponding genetic changes made to atleast one filamentous fungi background strain; obtaining a selection ofat least two genetic changes based at least in part upon a degree ofdissimilarity between the corresponding responsive performance measuresof the at least two genetic changes, wherein the degree of dissimilarityrelates to the degree to which the at least two genetic changes affecttheir corresponding responsive performance measures through differentbiological pathways; and designing genetic changes to an filamentousfungi background strain that include the selected genetic changes. Insome cases, the filamentous fungi background strain for which the atleast two selected genetic changes are designed is the same as the atleast one filamentous fungi background strain for which datarepresenting measured responsive performance was obtained.

In some embodiments, the present disclosure teaches HTP filamentousfungi strain improvement methods utilizing only a single type of geneticlibrary. For example, in some embodiments, the present disclosureteaches HTP filamentous fungi strain improvement methods utilizing onlySNP swap libraries. In other embodiments, the present disclosure teachesHTP filamentous fungi strain improvement methods utilizing only PRO swaplibraries. In some embodiments, the present disclosure teaches HTPfilamentous fungi strain improvement methods utilizing only STOP swaplibraries. In some embodiments, the present disclosure teaches HTPfilamentous fungi strain improvement methods utilizing only Start/StopCodon swap libraries.

In other embodiments, the present disclosure teaches HTP filamentousfungi strain improvement methods utilizing two or more types of geneticlibraries. For example, in some embodiments, the present disclosureteaches HTP filamentous fungi strain improvement methods combining SNPswap and PRO swap libraries. In some embodiments, the present disclosureteaches HTP filamentous fungi strain improvement methods combining SNPswap and STOP swap libraries. In some embodiments, the presentdisclosure teaches HTP filamentous fungi strain improvement methodscombining PRO swap and STOP swap libraries.

In other embodiments, the present disclosure teaches HTP filamentousfungi strain improvement methods utilizing multiple types of geneticlibraries. In some embodiments, the genetic libraries are combined toproduce combination mutations (e.g., promoter/terminator combinationladders applied to one or more genes). In yet other embodiments, the HTPfilamentous fungi strain improvement methods of the present disclosurecan be combined with one or more traditional strain improvement methods.

In some embodiments, the HTP filamentous fungi strain improvementmethods of the present disclosure result in an improved filamentousfungi host cell. That is, the present disclosure teaches methods ofimproving one or more filamentous fungi host cell properties. In someembodiments the improved filamentous fungi host cell property isselected from the group consisting of: volumetric productivity, specificproductivity, yield or titre, of a product of interest produced by thefilamentous fungi host cell. In some embodiments, the improvedfilamentous fungi host cell property is volumetric productivity. In someembodiments, the improved filamentous fungi host cell property isspecific productivity. In some embodiments, the improved filamentousfungi host cell property is yield.

In some embodiments, the HTP filamentous fungi strain improvementmethods of the present disclosure result in an filamentous fungi hostcell that exhibits a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 100%, 150%, 200%, 250%, 300% or more of an improvement inat least one filamentous fungi host cell property over a controlfilamentous fungi host cell that is not subjected to the HTP strainimprovements methods (e.g, an X % improvement in yield or productivityof a biomolecule of interest, incorporating any ranges and subrangestherebetween). In some embodiments, the HTP filamentous fungi strainimprovement methods of the present disclosure are selected from thegroup consisting of SNP swap, PRO swap, STOP swap, and combinationsthereof.

Thus, in some embodiments, the SNP swap methods of the presentdisclosure result in an filamentous fungi host cell that exhibits a 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%,200%, 250%, 300% or more of an improvement in at least one filamentousfungi host cell property over a control filamentous fungi host cell thatis not subjected to the SNP swap methods (e.g, an X % improvement inyield or productivity of a biomolecule of interest, incorporating anyranges and subranges therebetween).

Thus, in some embodiments, the PRO swap methods of the presentdisclosure result in an filamentous fungi host cell that exhibits a 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%,200%, 250%, 300% or more of an improvement in at least one filamentousfungi host cell property over a control filamentous fungi host cell thatis not subjected to the PRO swap methods (e.g, an X % improvement inyield or productivity of a biomolecule of interest, incorporating anyranges and subranges therebetween).

Thus, in some embodiments, the Terminator (STOP) swap methods of thepresent disclosure result in an filamentous fungi host cell thatexhibits a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 100%, 150%, 200%, 250%, 300% or more of an improvement in at leastone filamentous fungi host cell property over a control filamentousfungi host cell that is not subjected to the Terminator (STOP) swapmethods (e.g, an X % improvement in yield or productivity of abiomolecule of interest, incorporating any ranges and subrangestherebetween).

In one aspect, provided herein is a method for producing a filamentousfungal strain, the method comprising: a.) providing a plurality ofprotoplasts, wherein the protoplasts were prepared from a culture offilamentous fungal cells; b.) transforming the plurality of protoplastswith a first construct and a second construct, wherein the firstconstruct comprises a first polynucleotide flanked on both sides bynucleotides homologous to a first locus in the genome of the protoplastand the second construct comprises a second polynucleotide flanked onboth sides by nucleotides homologous to a second locus in the genome ofthe protoplast, wherein transformation results in integration of thefirst construct into the first locus and the second construct into thesecond locus by homologous recombination, wherein at least the secondlocus is a first selectable marker gene in the protoplast genome, andwherein the first polynucleotide comprises mutation and/or a geneticcontrol element; c.) purifying homokaryotic transformants by performingselection and counter-selection; and d.) growing the purifiedtransformants in media conducive to regeneration of the filamentousfungal cells. In some cases, the first construct is split into constructA and construct B, wherein construct A comprises a first portion of thefirst polynucleotide and nucleotides homologous to the first locus 5′ tothe first portion of the first polynucleotide, and wherein construct Bcomprises a second portion of the first polynucleotide and nucleotideshomologous to the first locus 3′ to the second portion of the firstpolynucleotide, wherein the first portion and the second portion of thefirst polynucleotide comprises overlapping complementary sequence. Insome cases, the second construct is split into construct A and constructB, wherein construct A comprises a first portion of the secondpolynucleotide and nucleotides homologous to the first locus 5′ to thefirst portion of the second polynucleotide, and wherein construct Bcomprises a second portion of the second polynucleotide and nucleotideshomologous to the first locus 3′ to the second portion of the secondpolynucleotide, wherein the first portion and the second portion of thesecond polynucleotide comprises overlapping complementary sequence. Insome cases, each protoplast from the plurality of protoplasts istransformed with a single first construct from a plurality of firstconstructs and a single second construct from a plurality of secondconstructs, wherein the first polynucleotide in each first constructfrom the plurality of first constructs comprises a different mutationand/or genetic control element; and wherein the second polynucleotide ineach second construct from the plurality of second constructs isidentical. In some cases, the method further comprises repeating stepsa-d to generate a library of filamentous fungal cells, wherein eachfilamentous fungal cell in the library comprises a first polynucleotidewith a different mutation and/or genetic control element. In some cases,the first polynucleotide encodes a target filamentous fungal gene or aheterologous gene. In some cases, the mutation is a single nucleotidepolymorphism. In some cases, the genetic control is a promoter sequenceand/or a terminator sequence. In some cases, the genetic control elementis a promoter sequence, wherein the promoter sequence is selected fromthe promoter sequences listed in Table 1. In some cases, the pluralityof protoplasts are distributed in wells of a microtiter plate. In somecases, steps a-d are performed in wells of a microtiter plate. In somecases, the microtiter plate is a 96 well, 384 well or 1536 wellmicrotiter plate. In some cases, the filamentous fungal cells areselected from Achlya, Acrenonium, Aspergillus, Aureobasidium,Bjerkandera, Ceriporiopsis. Cephalosporium, Chrysosporium, Cochliohohns,Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia,Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea,Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora,Penticillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor,Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces,Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma,Verticillium, Volvariella species or teleomorphs, or anamorphs, andsynonyms or taxonomic equivalents thereof. In some cases, thefilamentous fungal cells are Aspergillus niger. In some cases, thefilamentous fungal cells possess a non-mycelium forming phenotype. Insome cases, wherein the fungal cell possesses a non-functionalnon-homologous end joining (NHEJ) pathway. In some cases, the NHEJpathway is made non-functional by exposing the cell to an antibody, achemical inhibitor, a protein inhibitor, a physical inhibitor, a peptideinhibitor, or an anti-sense or RNAi molecule directed against acomponent of the NHEJ pathway. In some cases, the chemical inhibitor isW-7. In some cases, the first locus is for the target filamentous fungalgene. In some cases, the first locus is for a second selectable markergene in the protoplast genome. In some cases, the second selectablemarker gene is selected from an auxotrophic marker gene, a colorimetricmarker gene or a directional marker gene. In some cases, the firstselectable marker gene is selected from an auxotrophic marker gene, acolorimetric marker gene or a directional marker gene. In some cases,the second polynucleotide is selected from an auxotrophic marker gene, adirectional marker gene or an antibiotic resistance gene. In some cases,the colorimetric marker gene is an aygA gene. In some cases, theauxotrophic marker gene is selected from an argB gene, a trpC gene, apyrG gene, or a met3 gene. In some cases, the directional marker gene isselected from an acetamidase (amdS) gene, a nitrate reductase gene(niaD), or a sulphate permease (Sut B) gene. In some cases, theantibiotic resistance gene is a ble gene, wherein the ble gene confersresistance to pheomycin. In some cases, the first selectable marker geneis an aygA gene and the second polynucleotide is a pyrG gene. In somecases, the first selectable marker gene is a met3 gene, the secondselectable marker gene is an aygA gene and the second polynucleotide isa pyrG gene. In some cases, the plurality of protoplasts are prepared byremoving cell walls from the filamentous fungal cells in the culture offilamentous fungal cells, isolating the plurality of protoplasts; andresuspending the isolated plurality of protoplasts in a mixturecomprising dimethyl sulfoxide (DMSO), wherein the final concentration ofDMSO is 7% v/v or less. In some cases, the mixture is stored at at least−20° C. or −80° C. prior to performing steps a-d. In some cases, theculture is at least 1 liter in volume. In some cases, the culture isgrown for at least 12 hours prior to preparation of the protoplasts. Insome cases, the fungal culture is grown under conditions whereby atleast 70% of the protoplasts are smaller and contain fewer nuclei. Insome cases, removing the cell walls is performed by enzymatic digestion.In some cases, the enzymatic digestion is performed with mixture ofenzymes comprising a beta-glucanase and a polygalacturonase. In somecases, the method further comprises adding 40% v/v polyethylene glycol(PEG) to the mixture comprising DMSO prior to storing the protoplasts.In some cases, the PEG is added to a final concentration of 8% v/v orless. In some cases, steps a-d are automated.

In another aspect, provided herein is a method for preparing filamentousfungal cells for storage, the method comprising: preparing protoplastsfrom a fungal culture comprising filamentous fungal cells, wherein thepreparing the protoplasts comprises removing cell walls from thefilamentous fungal cells in the fungal culture; isolating theprotoplasts; and resuspending the isolated protoplasts in a mixturecomprising dimethyl sulfoxide (DMSO) at a final concentration of 7% v/vor less. In some cases, the mixture is stored at at least −20° C. or−80° C. In some cases, the fungal culture is at least 1 liter in volume.In some cases, the fungal culture is grown for at least 12 hours priorto preparation of the protoplasts. In some cases, the fungal culture isgrown under conditions whereby at least 70% of the protoplasts aresmaller and have fewer nuclei. In some cases, removing the cell walls isperformed by enzymatic digestion. In some cases, the enzymatic digestionis performed with mixture of enzymes comprising a beta-glucanase and apolygalacturonase. In some cases, the method further comprises adding40% v/v polyethylene glycol (PEG) to the mixture comprising DMSO priorto storing the protoplasts. In some cases, the PEG is added to a finalconcentration of 8% v/v or less. In some cases, the method furthercomprises distributing the protoplasts into microtiter plates prior tostoring the protoplasts. In some cases, the filamentous fungal cells inthe fungal culture possess a non-mycelium forming phenotype. In somecases, the filamentous fungal cells in the fungal culture are selectedfrom Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus. Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora(e.g., Myceliophthora thermophila), Mucor, Neurospora, Penicillium,Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus,Schizophyllum, Scylalidium, Sporotrichum, Talaromyces, Thermoascus,Thielavia, Tramates, Tolypocladium, Trichoderma, Verticillium,Volvariella species or teleomorphs, or anamorphs, and synonyms ortaxonomic equivalents thereof. In some cases, the filamentous fungalcells in the fungal culture are Aspergillus niger or teleomorphs oranamorphs thereof.

In yet another aspect, provided herein is a system for generating afungal production strain, the system comprising: one or more processors;and one or more memories operatively coupled to at least one of the oneor more processors and having instructions stored thereon that, whenexecuted by at least one of the one or more processors, cause the systemto: a.) transform a plurality of protoplasts derived from culture offilamentous fungal cells with a first construct and a second construct,wherein the first construct comprises a first polynucleotide flanked onboth sides by nucleotides homologous to a first locus in the genome ofthe protoplast and the second construct comprises a secondpolynucleotide flanked on both sides by nucleotides homologous to asecond locus in the genome of the protoplast, wherein transformationresults in integration of the first construct into the first locus andthe second construct into the second locus by homologous recombination,wherein at least the second locus is a first selectable marker gene inthe protoplast genome, and wherein the first polynucleotide comprises amutation and/or a genetic control element; b.) purifying homokaryotictransformants by performing selection and counter-selection; and c.)growing the purified transformants in media conducive to regeneration ofthe filamentous fungal cells. In some cases, the first construct issplit into construct A and construct B, wherein construct A comprises afirst portion of the first polynucleotide and nucleotides homologous tothe first locus 5′ to the first portion of the first polynucleotide, andwherein construct B comprises a second portion of the firstpolynucleotide and nucleotides homologous to the first locus 3′ to thesecond portion of the first polynucleotide, wherein the first portionand the second portion of the first polynucleotide comprises overlappingcomplementary sequence. In some cases, the second construct is splitinto construct A and construct B, wherein construct A comprises a firstportion of the second polynucleotide and nucleotides homologous to thefirst locus 5′ to the first portion of the second polynucleotide, andwherein construct B comprises a second portion of the secondpolynucleotide and nucleotides homologous to the first locus 3′ to thesecond portion of the second polynucleotide, wherein the first portionand the second portion of the second polynucleotide comprisesoverlapping complementary sequence. In some cases, each protoplast fromthe plurality of protoplasts is transformed with a single firstconstruct from a plurality of first constructs and a single secondconstruct from a plurality of second constructs, wherein the firstpolynucleotide in each first construct from the plurality of firstconstructs comprises a different mutation and/or genetic controlelement; and wherein the second polynucleotide in each second constructfrom the plurality of second constructs is identical. In some cases, thesystem further comprises repeating steps a-c to generate a library offilamentous fungal cells, wherein each filamentous fungal cell in thelibrary comprises a first polynucleotide with a different mutationand/or genetic control element. In some cases, the mutation is a singlenucleotide polymorphism. In some cases, the genetic control is apromoter sequence and/or a terminator sequence. In some cases, thegenetic control element is a promoter sequence, wherein the promotersequence is selected from the promoter sequences listed in Table 1. Insome cases, steps a-c are performed in wells of a microtiter plate. Insome cases, the microtiter plate is a 96 well, 384 well or 1536 wellmicrotiter plate. In some cases, the filamentous fungal cells areselected from Achlya, Acremonium, Aspergillus, Aureobasidium,Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus,Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia,Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea,Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora,Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor,Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces,Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma,Verticillium, Volvariella species or teleomorphs, or anamorphs, andsynonyms or taxonomic equivalents thereof. In some cases, thefilamentous fungal cells are Aspergillus niger. In some cases, thefilamentous fungal cells possess a non-mycelium forming phenotype. Insome cases, the fungal cell possesses a non-functional non-homologousend joining pathway. In some cases, the NHEJ pathway is madenon-functional by exposing the cell to an antibody, a chemicalinhibitor, a protein inhibitor, a physical inhibitor, a peptideinhibitor, or an anti-sense or RNAi molecule directed against acomponent of the NHEJ pathway. In some cases, the chemical inhibitor isW-7. In some cases, the first locus is for the target filamentous fungalgene. In some cases, the first locus is for a second selectable markergene in the protoplast genome. In some cases, the second selectablemarker gene is selected from an auxotrophic marker gene, a colorimetricmarker gene or a directional marker gene. In some cases, the firstselectable marker gene is selected from an auxotrophic marker gene, acolorimetric marker gene or a directional marker gene. In some cases,the second polynucleotide is selected from an auxotrophic marker gene, adirectional marker gene or an antibiotic resistance gene. In some cases,the colorimetric marker gene is an aygA gene. In some cases, theauxotrophic marker gene is selected from an argB gene, a trpC gene, apyrG gene, or a met3 gene. In some cases, the directional marker gene isselected from an acetamidase (amdS) gene, a nitrate reductase gene(nlaD), or a sulphate permease (Sut B) gene. In some cases, theantibiotic resistance gene is a ble gene, wherein the ble gene confersresistance to pheomycin. In some cases, the first selectable marker geneis an aygA gene and the second polynucleotide is a pyrG gene. In somecases, the first selectable marker gene is a met3 gene, the secondselectable marker gene is an aygA gene and the second polynucleotide isa pyrG gene. In some cases, the plurality of protoplasts are prepared byremoving cell walls from the filamentous fungal cells in the culture offilamentous fungal cells; isolating the plurality of protoplasts; andresuspending the isolated plurality of protoplasts in a mixturecomprising dimethyl sulfoxide (DMSO) at a final concentration of 7% v/vor less. In some cases, the mixture is stored at at least −20° C. or−80° C. prior to performing steps a-c. In some cases, the culture is atleast 1 liter in volume. In some cases, the culture is grown for atleast 12 hours prior to preparation of the protoplasts. In some cases,the fungal culture is grown under conditions whereby at least 70% of theprotoplasts are smaller and have fewer nuclei. In some cases, removingthe cell walls is performed by enzymatic digestion. In some cases, theenzymatic digestion is performed with mixture of enzymes comprising abeta-glucanase and a polygalacturonase. In some cases, the systemfurther comprises adding 40% v/v polyethylene glycol (PEG) to themixture comprising DMSO prior to storing the protoplasts. In some cases,the PEG is added to a final concentration of 8% v/v or less.

In yet another aspect, provided herein is a method for isolating clonalpopulations derived from single fungal spores, the method comprising:(a) providing a plurality of fungal spores in a liquid suspension,wherein the plurality of fungal spores were derived from a fungalstrain; (b) dispensing a discrete volume of the liquid suspension to anindividual reaction area in a substrate comprising a plurality ofreaction areas, wherein each reaction area in the plurality of reactionareas comprises growth media, wherein the dispensing results in aprobability that at least 75% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores; (c) culturing the dispensed single viable fungal spores in thereaction areas comprising growth media; and (d) selecting clonalpopulations growing in the reaction areas, thereby isolating clonalpopulations derived from single fungal spores. In some cases, the methodfurther comprises screening the discrete volumes for the presence orabsence of a single fungal spore in the discrete volumes, wherein onlythe discrete volumes containing a single fungal spore are selected forstep (b). In some cases, the dispensing results in a probability that atleast 80% of the individual reaction areas contain no more than a singleviable fungal spore from the plurality of fungal spores. In some cases,the dispensing results in a probability that at least 90% of theindividual reaction areas contain no more than a single viable fungalspore from the plurality of fungal spores. In some cases, the dispensingresults in a probability that at least 95% of the individual reactionareas contain no more than a single viable fungal spore from theplurality of fungal spores. In some cases, the dispensing results in aprobability that at least 99/o of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores. In some cases, the dispensing results in a probability thatsubstantially all of the individual reaction areas contain no more thana single viable fungal spore from the plurality of fungal spores. Insome cases, the screening the discrete volumes entails opticallydistinguishing the presence or absence of a single fungal spore in thediscrete volumes. In some cases, the screening is performed using amicrofluidic device capable of optically distinguishing the presence orabsence of a single fungal spore in the discrete volumes. In some cases,the reaction areas are present in a microtiter plate. In some cases, themicrotiter plate contains 96 wells, 384 wells or 1536 wells. In somecases, the fungal strain is a filamentous fungal strain. In some cases,the filamentous fungal strain is selected from Achlya, Acremonium,Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium,Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus,Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella,Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthorathermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia,Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof. In some cases, the filamentous fungal strain is Aspergillusniger or teleomorphs or anamorphs thereof. In some cases, thefilamentous fungal strain possess a non-mycelium, pellet morphology. Insome cases, the filamentous fungal strain expresses a mutant form of anortholog of the S. cerevisiae SLN1 gene. In some cases, the filamentousfungal strain is A. niger and a nucleic sequence of the mutant form ofthe A. niger ortholog of the S. cerevisiae SLN1 gene is SEQ ID NO: 13.In some cases, the mutant form of the orthologue of the S. cerevisiaeSLN1 gene (e.g., the A. niger orthologue) is operably linked to apromoter sequence selected from SEQ ID NO: 1 or 2. In some cases, thefungal strain possesses a genetic perturbation. In some cases, thegenetic perturbation is selected from single nucleotide polymorphisms,DNA insertions, DNA deletions or any combination thereof. In some cases,the genetic perturbation is introduced into protoplasts derived from thefungal strain via transforming the protoplasts with a ribonucleoproteincomplex (RNP-complex). In some cases, the RNP-complex comprises an RNAguided endonuclease complexed with a guide RNA (gRNA). In some cases,the RNA guided endonuclease is a Class 2 CRISPR-Cas System RNA guidedendonuclease. In some cases, the Class 2 CRISPR-Cas system RNA guidedendonuclease is a Type II, Type V or Type VI RNA guided endonuclease. Insome cases, the Class 2 CRISPR-Cas system RNA guided endonuclease isselected from Cas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a,Cas13b, Cas13c or homologs, orthologs, mutants, variants or modifiedversions thereof. In some cases, the Class 2 CRISPR-Cas system RNAguided endonuclease is Cas9 or homologs, orthologs or paralogs thereof.In some cases, the gRNA is a CRISPR RNA (crRNA) alone or annealed to atransactivating CRISPR RNA (tracrRNA). In some cases, the gRNA is asingle guide RNA (sgRNA) comprising a tracrRNA and a crRNA. In somecases, the crRNA comprises a guide sequence complementary to a targetgene within the genome of the fungal strain, wherein introduction of theRNP-complex into the protoplasts facilitates introduction of the geneticperturbation into the target gene. In some cases, the geneticperturbation of the target gene is facilitated by cleavage of the targetgene by the RNP-complex to generate DNA ends in the target gene followedby non-homologous end joining of the DNA ends in the target gene by thenon-homologous end joining (NHEJ) pathway. In some cases, the methodfurther comprises co-transforming a donor DNA comprising a mutatedversion of the target gene, wherein the mutated version of the targetgene is flanked on both sides by nucleotides homologous to the targetgene locus. In some cases, the genetic perturbation of the target geneis facilitated by cleavage of the target gene by the RNP-complex togenerate DNA ends in the target gene followed by replacement of thetarget gene with the donor DNA via homologous recombination. In somecases, step (b) further comprises co-transforming a vector comprising aselectable marker. In some cases, the selectable marker is used duringstep (d) to select clonal populations derived from transformationcompetent fungal strains. In some cases, the genetic perturbation isintroduced into protoplasts derived from the fungal strain bytransforming the plurality of protoplasts with a first construct and asecond construct, wherein the first construct comprises a firstpolynucleotide flanked on both sides by nucleotides homologous to afirst locus in the genome of the protoplast and the second constructcomprises a second polynucleotide flanked on both sides by nucleotideshomologous to a second locus in the genome of the protoplast, whereinthe transformation results in integration of the first construct intothe first locus and the second construct into the second locus byhomologous recombination, wherein at least the second locus is a firstselectable marker gene in the protoplast genome, and wherein the firstpolynucleotide comprises the genetic perturbation. In some cases, theselectable marker gene is used during step (d) to facilitate selectionof clonal populations derived from fungal strains comprising the geneticperturbation. In some cases, the fungal strain possesses anon-functional non-homologous end joining (NHEJ) pathway. In some cases,the NHEJ pathway is made non-functional by exposing the fungal strain toan antibody, a chemical inhibitor, a protein inhibitor, a physicalinhibitor, a peptide inhibitor, or an anti-sense or RNAi moleculedirected against a component of the NHEJ pathway. In some cases, thechemical inhibitor is W-7.

In another aspect, provided herein is a method for isolating clonalpopulations derived from single fungal spores, the method comprising.(a) providing a plurality of fungal spores in a liquid suspension,wherein the plurality of fungal spores were derived from a fungalstrain; (b) diluting the liquid suspension, wherein the dilution is alimiting dilution; (c) dispensing a discrete volume of the dilution toan individual reaction area in a substrate comprising a plurality ofreaction areas, wherein each reaction area in the plurality of reactionareas comprises growth media, wherein the limiting dilution results in aprobability that the discrete volume of the dilution dispensed to eachreaction area contains either one or no viable spore follows a PoissonDistribution, whereby greater than 90% b of the reaction areas in theplurality of reaction areas contain no viable spores and greater than90% of reaction areas that contain one or more viable spores containonly a single viable spore; (d) culturing the dispensed single viablefungal spores in the reaction areas comprising growth media; and (e)selecting clonal populations growing in the reaction areas, therebyisolating clonal populations derived from single fungal spores. In somecases, the reaction areas are present in a microtiter plate. In somecases, the microtiter plate contains 96 wells, 384 wells or 1536 wells.In some cases, the fungal strain is a filamentous fungal strain. In somecases, the filamentous fungal strain is selected from Achlya,Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria,Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium,Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g.,Myceliophthora thermophila), Mucor, Neurospora, Penicillium, Podospora,Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof. In some cases, the filamentous fungal strain is Aspergillusniger or teleomorphs or anamorphs thereof. In some cases, thefilamentous fungal strain possess a non-mycelium, pellet morphology. Insome cases, the filamentous fungal strain expresses a mutant form of anortholog of the S. cerevisiae SLN1 gene. In some cases, the filamentousfungal strain is A. niger and a nucleic sequence of the mutant form ofthe A. niger orthologue of the S. cerevisiae SLN1 gene is SEQ ID NO: 13.In some cases, the mutant form of the orthologue of the S. cerevisiaeSLN1 gene (e.g., the A. niger orthologue) is operably linked to apromoter sequence selected from SEQ ID NO: 1 or 2. In some cases, thefungal strain possesses a genetic perturbation. In some cases, thegenetic perturbation is selected from single nucleotide polymorphisms,DNA insertions, DNA deletions or any combination thereof. In some cases,the genetic perturbation is introduced into protoplasts derived from thefungal strain via transforming the protoplasts with a ribonucleoproteincomplex (RNP-complex). In some cases, the RNP-complex comprises an RNAguided endonuclease complexed with a guide RNA (gRNA). In some cases,the RNA guided endonuclease is a Class 2 CRISPR-Cas System RNA guidedendonuclease. In some cases, the Class 2 CRISPR-Cas system RNA guidedendonuclease is a Type II, Type V or Type VI RNA guided endonuclease. Insome cases, the Class 2 CRISPR-Cas system RNA guided endonuclease isselected from Cas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a,Cas13b, Cas13c or homologs, orthologs, mutants, variants or modifiedversions thereof. In some cases, the Class 2 CRISPR-Cas system RNAguided endonuclease is Cas9 or homologs, orthologs or paralogs thereof.In some cases, the gRNA is a CRISPR RNA (crRNA) alone or annealed to atransactivating CRISPR RNA (tracrRNA). In some cases, the gRNA is asingle guide RNA (sgRNA) comprising a tracrRNA and a crRNA. In somecases, the crRNA comprises a guide sequence complementary to a targetgene within the genome of the fungal strain, wherein introduction of theRNP-complex into the protoplasts facilitates introduction of the geneticperturbation into the target gene. In some cases, the geneticperturbation of the target gene is facilitated by cleavage of the targetgene by the RNP-complex to generate DNA ends in the target gene followedby non-homologous end joining of the DNA ends in the target gene by thenon-homologous end joining (NHEJ) pathway. In some cases, the methodfurther comprises co-transforming a donor DNA comprising a mutatedversion of the target gene, wherein the mutated version of the targetgene is flanked on both sides by nucleotides homologous to the targetgene locus. In some cases, the genetic perturbation of the target geneis facilitated by cleavage of the target gene by the RNP-complex togenerate DNA ends in the target gene followed by replacement of thetarget gene with the donor DNA via homologous recombination. In somecases, step (b) further comprises co-transforming a vector comprising aselectable marker. In some cases, the selectable marker is used duringstep (d) to select clonal populations derived from transformationcompetent fungal strains. In some cases, the genetic perturbation isintroduced into protoplasts derived from the fungal strain bytransforming the plurality of protoplasts with a first construct and asecond construct, wherein the first construct comprises a firstpolynucleotide flanked on both sides by nucleotides homologous to afirst locus in the genome of the protoplast and the second constructcomprises a second polynucleotide flanked on both sides by nucleotideshomologous to a second locus in the genome of the protoplast, whereinthe transformation results in integration of the first construct intothe first locus and the second construct into the second locus byhomologous recombination, wherein at least the second locus is a firstselectable marker gene in the protoplast genome, and wherein the firstpolynucleotide comprises the genetic perturbation. In some cases, theselectable marker gene is used during step (d) to facilitate selectionof clonal populations derived from fungal strains comprising the geneticperturbation. In some cases, the fungal strain possesses anon-functional non-homologous end joining (NHEJ) pathway. In some cases,the NHEJ pathway is made non-functional by exposing the fungal strain toan antibody, a chemical inhibitor, a protein inhibitor, a physicalinhibitor, a peptide inhibitor, or an anti-sense or RNAi moleculedirected against a component of the NHEJ pathway. In some cases, thechemical inhibitor is W-7.

In one aspect, provided herein is a method for producing a filamentousfungal strain, the method comprising: a.) providing a plurality ofprotoplasts, wherein the plurality of protoplasts were prepared from aculture of a parent filamentous fungal strain; b.) transforming eachprotoplast from the plurality of protoplasts with a ribonucleoproteincomplex (RNP-complex); and c.) selecting and screening individualfilamentous fungal strains derived from the transformed protoplasts forphenotypic performance improvements over the parent filamentous fungalstrain, thereby identifying genetic perturbations in the genome of theselected individual filamentous fungal strains that confer phenotypicperformance improvements. In some cases, the genetic perturbations areselected from single nucleotide polymorphisms, DNA insertions, DNAdeletions or any combination thereof. In some cases, the RNP-complexcomprises an RNA guided endonuclease complexed with a guide RNA (gRNA).In some cases, the RNA guided endonuclease is a Class 2 CRISPR-CasSystem RNA guided endonuclease. In some cases, the Class 2 CRISPR-Cassystem RNA guided endonuclease is a Type II, Type V or Type VI RNAguided endonuclease. In some cases, the Class 2 CRISPR-Cas system RNAguided endonuclease is selected from Cas9, Cas12a, Cas12b, Cas12c,Cas12d, Cas12e, Cas13a, Cas13b, Cas13c or homologs, orthologs, mutants,variants or modified versions thereof. In some cases, the Class 2CRISPR-Cas system RNA guided endonuclease is Cas9 or homologs, orthologsor paralogs thereof. In some cases, the gRNA is a CRISPR RNA (crRNA)alone or annealed to a transactivating CRISPR RNA (tracrRNA). In somecases, the gRNA is a single guide RNA (sgRNA) comprising a tracrRNA anda crRNA. In some cases, the crRNA comprises a guide sequence that iscomplementary to a target gene within the genome of the parentfilamentous fungal strain, wherein introduction of the RNP-complexperturbs the target gene in the protoplasts. In some cases, theperturbation of the target gene is facilitated by cleavage of the targetgene by the RNP-complex to generate DNA ends in the target gene followedby non-homologous end joining of the DNA ends in the target gene by thenon-homologous end joining (NHEJ) pathway. In some cases, step (b)further comprises co-transforming a donor DNA comprising a mutatedversion of the target gene, wherein the mutated version of the targetgene is flanked on both sides by nucleotides homologous to the targetgene locus. In some cases, the perturbation of the target gene isfacilitated by cleavage of the target gene by the RNP-complex togenerate DNA ends in the target gene followed by replacement of thetarget gene with the donor DNA via homologous recombination. In somecases, step (b) further comprises co-transforming a vector comprising aselectable marker. In some cases, the selectable marker is used duringstep (c) to select transformation competent individual filamentousfungal strains for subsequent screening for phenotypic performanceimprovements over the parent filamentous fungal strain. In some cases,the parent filamentous fungal strain possesses a non-functionalnon-homologous end joining (NHEJ) pathway. In some cases, the NHEJpathway is made non-functional by exposing the cell to an antibody, achemical inhibitor, a protein inhibitor, a physical inhibitor, a peptideinhibitor, or an anti-sense or RNAi molecule directed against acomponent of the NHEJ pathway. In some cases, the chemical inhibitor isW-7. In some cases, the phenotypic performance improvement of thefilamentous fungal strain comprises at least a 10% increase in ameasured phenotypic variable for a product of interest compared to thephenotypic performance of the parent filamentous fungal strain. In somecases, the phenotypic performance improvement of the filamentous fungalstrain comprises at least a one-fold increase in a measured phenotypicvariable for a product of interest compared to the phenotypicperformance of the parent filamentous fungal strain. In some cases, themeasured phenotypic variable is selected from the group consisting of:volumetric productivity of the product of interest, specificproductivity of the product of interest, yield of the product ofinterest, titer of the product of interest, and combinations thereof. Insome cases, the measured phenotypic variable is increased or moreefficient production of the product of interest. In some cases, theproduct of interest is selected from the group consisting of: a smallmolecule, enzyme, peptide, amino acid, organic acid, synthetic compound,fuel, alcohol, primary extracellular metabolite, secondary extracellularmetabolite, intracellular component molecule, and combinations thereof.In some cases, the parent filamentous fungal strain is selected fromAchlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium. Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora(e.g., Myceliophthora thermophila), Mucor, Neurospora, Penicillium,Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus,Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus,Thielavia, Tramates, Tolypocladium, Trichoderma, Verticillium,Volvariella species or teleomorphs, or anamorphs, and synonyms ortaxonomic equivalents thereof. In some cases, the filamentous fungalstrain is Aspergillus niger or teleomorphs or anamorphs thereof. In somecases, the filamentous fungal strain possess a non-mycelium, pelletmorphology. In some cases, the filamentous fungal strain expresses amutant form of an orthologue of the S. cerevisiae SLN1 gene. In somecases, the filamentous fungal strain is A. niger and a nucleic sequenceof the mutant form of the A. niger orthologue of the S. cerevisiae SLN1gene is SEQ ID NO: 13. In some cases, the mutant form of the orthologueof the S. cerevisiae SLN1 gene (e.g., the A. niger orthologue) isoperably linked to a promoter sequence selected from SEQ ID NO: 1 or 2.In some cases, the method further comprises generating isolated clonalpopulations derived from the individual filamentous fungal strains priorto step (c). In some cases, the isolating comprises: (i) inducing thetransformed protoplasts to produce a plurality of fungal spores, whereineach fungal spore form the plurality is derived from a singletransformed protoplast; (ii) resuspending the plurality of fungal sporesderived from a single transformed protoplast in a liquid to generate aliquid suspension; (iii) dispensing a discrete volume of the liquidsuspension to an individual reaction area in a substrate comprising aplurality of reaction areas, wherein each reaction area in the pluralityof reaction areas comprises growth media, wherein the dispensing resultsin a probability that at least 75% of the individual reaction areascontain no more than a single viable fungal spore from the plurality offungal spores; and (iv) culturing the dispensed single viable fungalspores in the reaction areas comprising growth media, thereby generatingisolated clonal populations derived from the individual filamentousfungal strains. In some cases, the method further comprises screeningthe discrete volumes for the presence or absence of a single fungalspore in the discrete volumes, wherein only the discrete volumescontaining a single fungal spore are selected for step (iii). In somecases, the screening the discrete volumes entails opticallydistinguishing the presence or absence of a single fungal spore in thediscrete volumes. In some cases, the screening is performed using amicrofluidic device capable of optically distinguishing the presence orabsence of a single fungal spore in the discrete volumes. In some cases,the dispensing results in a probability that at least 80% of theindividual reaction areas contain no more than an single viable fungalspore from the plurality of fungal spores. In some cases, the dispensingresults in a probability that at least 90% of the individual reactionareas contain no more than a single viable fungal spore from theplurality of fungal spores. In some cases, the dispensing results in aprobability that at least 95% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores. In some cases, the dispensing results in a probability that atleast 99% of the individual reaction areas contain no more than a singleviable fungal spore from the plurality of fungal spores. In some cases,the dispensing results in a probability that substantially all of theindividual reaction areas contain no more than a single viable fungalspore from the plurality of fungal spores In some cases, the isolatingcomprises: (i) inducing the transformed protoplasts to produce aplurality of fungal spores, wherein each fungal spore form the pluralityis derived from a single transformed protoplast; (ii) resuspending theplurality of fungal spores derived from a single transformed protoplastin a liquid to generate a liquid suspension; (iii) diluting the liquidsuspension, wherein the dilution is a limiting dilution; (iv) dispensinga discrete volume of the dilution to an individual reaction area in asubstrate comprising a plurality of reaction areas, wherein eachreaction area in the plurality of reaction areas comprises growth media,wherein the limiting dilution results in a probability that the discretevolume of the dilution dispensed to each reaction area contains eitherone or no viable spore follows a Poisson Distribution, whereby greaterthan 90% of the reaction areas in the plurality of reaction areascontain no viable spores and greater than 90% of reaction areas thatcontain one or more viable spores contain only a single viable spore;(v) culturing the dispensed single viable fungal spores in the reactionareas comprising growth media; and (vi) selecting clonal populationsgrowing in the reaction areas, thereby isolating clonal populationsderived from single fungal spores. In some cases, the reaction areas arepresent in a microtiter plate. In some cases, the microtiter platecontains 96 wells, 384 wells or 1536 wells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a DNA recombination method of the present disclosure forincreasing variation in diversity pools. DNA sections, such as genomeregions from related species, can be cut via physical orenzymatic/chemical means. The cut DNA regions are melted and allowed toreanneal, such that overlapping genetic regions prime polymeraseextension reactions. Subsequent melting/extension reactions are carriedout until products are reassembled into chimeric DNA, comprisingelements from one or more starting sequences.

FIG. 2 outlines methods of the present disclosure for generating newhost filamentous fungal strains with selected sequence modifications(e.g., 100 SNPs to swap). Briefly, the method comprises (1) desired DNAinserts are designed and generated using any of the methods providedherein, (2) DNA inserts are cloned into transformation constructs, (3)completed constructs are transferred into desired strains (e.g., base orproduction strains), where they are integrated into the host straingenome, and (4) selection markers and other unwanted DNA elements arelooped out of the host strain. Each DNA assembly step may involveadditional quality control (QC) steps, such as cloning constructs intofilamentous fungal cells for amplification and sequencing. Thetransformation step can be preceded by a protoplasting step. Theprotoplasting can be performed using any protoplasting method known inthe art. In one embodiment, protoplasting of the filamentous fungal hostcells is performed using the method provided herein. In one embodiment,protoplasts are generated from the filamentous fungal host cells priorto transformation.

FIG. 3 is a representation of how SNPs are targeted to a specific locusin filamentous fungi using a split marker system. The marker gene (pyrGin this example) is amplified into two components that are unable tocomplement the mutation in the target strain without homologousrecombination, which restores gene function. Flanking these fragments isa direct repeat of DNA that each of which contains the SNPs to betargeted to the locus. Non-repeat DNA sequence on each constructfacilitates proper integration through native homologous recombinationpathways. These constructs are placed into the target strains duringstep 2 of FIG. 20B.

FIG. 4 illustrates that the direct repeats flanking the marker gene areunstable and will result in marker removal through homologousrecombination between the direct repeats. Essentially, the loop-out isfacilitated by direct repeats that were incorporated into thetransforming DNA. Cells counter selected for the selection markercontain deletions of the loop DNA flanked by the direct repeat regions.

FIG. 5 depicts an embodiment of the filamentous fungal strainimprovement process of the present disclosure. Host strain sequencescontaining genetic modifications (Genetic Design) are tested for strainperformance improvements in various strain backgrounds (Strain Build).Strains exhibiting beneficial mutations are analyzed (Hit ID andAnalysis) and the data is stored in libraries for further analysis(e.g., SNP swap libraries, PRO swap libraries, and combinations thereof,among others). Selection rules of the present disclosure generate newproposed filamentous fungal host strain sequences based on the predictedeffect of combining elements from one or more libraries for additionaliterative analysis.

FIG. 6A-6B depicts the DNA assembly, transformation, and filamentousfungal strain screening steps of one of the embodiments of the presentdisclosure. FIG. 6A depicts the steps for building DNA fragments,cloning said DNA fragments, transforming said fragments into hostfilamentous fungal strains, and looping out selection sequences throughcounter selection. FIG. 6B depicts the steps for high-throughputculturing, screening, and evaluation of selected filamentous fungal hoststrains. This figure also depicts the optional steps of culturing,screening, and evaluating selected filamentous fungal strains in culturetanks.

FIG. 7 depicts one embodiment of the automated system of the presentdisclosure. The present disclosure teaches use of automated roboticsystems with various modules capable of cloning, transforming,culturing, screening and/or sequencing host filamentous fungus

FIG. 8 depicts an overview of an embodiment of the filamentous fungalstrain improvement program of the present disclosure.

FIG. 9 depicts a first-round SNP swapping experiment according to themethods of the present disclosure. (1) all the SNPs from C will beindividually and/or combinatorially cloned into the base A strain (“waveup” A to C). (2) all the SNPs from C will be individually and/orcombinatorially removed from the commercial strain C (“wave down” C toA). (3) all the SNPs from B will be individually and/or combinatoriallycloned into the base A strain (wave up A to B). (4) all the SNPs from Bwill be individually and/or combinatorially removed from the commercialstrain B (wave down B to A). (5) all the SNPs unique to C will beindividually and/or combinatorially cloned into the commercial B strain(wave up B to C). (6) all the SNPs unique to C will be individuallyand/or combinatorially removed from the commercial strain C (wave down Cto B).

FIG. 10 depicts the different available approaches to promoter swapping.In particular, a promoter swap design for a gene with an annotatedpromoter is shown.

FIG. 11 depicts the DNA assembly and transformation steps of one of theembodiments of the present disclosure. The flow chart depicts the stepsfor building DNA fragments, cloning said DNA fragments, transformingsaid DNA fragments into host filamentous fungal strains, and looping outselection sequences through counter selection.

FIG. 12 depicts the steps for high-throughput culturing, screening, andevaluation of selected host filamentous fungal strains. This figure alsodepicts the optional steps of culturing, screening, and evaluatingselected filamentous fungal strains in culture tanks.

FIG. 13 depicts expression profiles of illustrative promoters exhibitinga range of regulatory expression, according to the promoter ladders ofthe present disclosure. Promoter A expression peaks immediately uponaddition of a selected substrate, but quickly returns to undetectablelevels as the concentration of the substrate is reduced. Promoter Bexpression peaks immediately upon addition of the selected substrate andlowers slowly back to undetectable levels together with thecorresponding reduction in substrate. Promoter C expression peaks uponaddition of the selected substrate, and remains highly expressedthroughout the culture, even after the substrate has dissipated.

FIG. 14 diagrams an embodiment of LIMS system of the present disclosurefor filamentous fungal strain improvement.

FIG. 15 diagrams a cloud computing implementation of embodiments of theLIMS system of the present disclosure.

FIG. 16 depicts an embodiment of the iterative predictive strain designworkflow of the present disclosure.

FIG. 17 diagrams an embodiment of a computer system, according toembodiments of the present disclosure.

FIG. 18 depicts the workflow associated with the DNA assembly accordingto one embodiment of the present disclosure. This process is divided upinto 4 stages: parts generation, plasmid/construct assembly,plasmid/construct QC, and plasmid/construct preparation fortransformation. During parts generation, oligos designed by LaboratoryInformation Management System (LIMS) are ordered from an oligosequencing vendor and used to amplify the target sequences from the hostorganism via PCR. These PCR parts are cleaned to remove contaminants andassessed for success by fragment analysis, in silico quality controlcomparison of observed to theoretical fragment sizes, and DNAquantification. The parts are transformed into yeast along with anassembly vector and assembled into plasmids via homologousrecombination. Assembled plasmids are isolated from yeast andtransformed into E. coli for subsequent assembly quality control andamplification. During plasmid assembly quality control, severalreplicates of each plasmid are isolated, amplified using Rolling CircleAmplification (RCA), and assessed for correct assembly by enzymaticdigest and fragment analysis. Correctly assembled plasmids identifiedduring the QC process are hit picked to generate permanent stocks andthe specific gene construct including any flanking sequences necessaryto facilitate genome integration are then PCR amplified from the plasmidto generate linear DNA fragments that are quantified prior totransformation into the target host organism (e.g., filamentous fungalhost cell). As an alternative to generating plasmids as described above,fusion PCR can be used to generate specific gene constructs includingany flanking sequences necessary to facilitate genome integration in afilamentous fungal host cell.

FIG. 19 is a flowchart illustrating the consideration of epistaticeffects in the selection of mutations for the design of a microbialstrain, according to embodiments of the disclosure.

FIG. 20A depicts a general outline for the automated transformation,screening and purification of homokaryotic protoplasts provided hereinand described in Example 1. FIG. 20B illustrates steps in the process ofSNP/PRO/STOP swapping in filamentous fungi. FIG. 20C illustrates stepsin the process of screening the transformants for proper integrationusing any of the swapping methods provided throughout this disclosure.

FIG. 21 depicts screening of A. niger mutant strain cotransformantsutilizing the argB marker by observing growth of A. niger mutant strainson minimal media with and without arginine following automatedtransformation and screening as described in Example 2. Successfulco-transformation resulted in disruption of the argB gene and no growthon minimal media.

FIG. 22 depicts characterization of heterokaryons/homokaryons. Inparticular, this figure illustrates screening of A. niger mutant strainsutilizing the aygA colorimetric gene marker by observing growth of A.niger mutant strains on minimal media following automated transformationand screening as described in Example 3. Colonies derived fromhomokaryotic protoplasts were pure yellow in color and lacked blackspores.

FIG. 23A-B depicts the results of A. niger transformation and validationaccording to the methods of the present disclosure. FIG. 23A is apicture of a 96-well media plate of A. niger transformants. Transformedcultures comprise a mutation in the aygA, which causes the cells toappear lighter yellow instead of black (transformed wells are circled inwhite). FIG. 23B depicts the results of next generation sequencing oftransformed A. niger mutants. The X-axis represents the target DNA'ssequence identity with the untransformed parent strain. The Y-axisrepresents the target DNA's sequence identity with the expectedmutation. Data points towards the bottom right of the chart exhibit highsimilarity with the parent strain, and low similarity with the expectedtransformed sequences. Data points towards the top left of the chartexhibit high similarity to expected transformed sequences and lowidentity with parent strain. Data points in the middle likely representheterokaryons with multiple nuclei.

FIG. 24 depicts a SNP swap implementation in A. niger. The left side ofFIG. 24 illustrates the designed genetic edits for each SNP of the SNPswap. The FIG. 24 further illustrates the cotransformation in which thepyrG gene is introduced into the locus for the aygA wild type gene. Theright side of FIG. 24 shows two pictures of the 96-well media plates forscreening the A. niger transformants. Light yellow colonies representtransformants in which the aygA gene has been successfully disrupted.The A. niger strain used to build the mutant strains depicted withinFIG. 24 were strains with reduced NHEJ pathway activity.

FIG. 25 depicts a quality control (QC) chart identifying successful A.niger mutant transformants (top box) based on next generation sequencingresults. Overall 29.2% of yellow colonies selected from the cultureplates exhibit the expected SNP genetic change.

FIG. 26 depicts the results of next generation sequencing of transformedA. niger mutants. The X-axis represents the target DNA's sequenceidentity with the untransformed parent strain. The Y-axis represents thetarget DNA's sequence identity with the expected mutation. Data pointstowards the bottom right of the chart exhibit high similarity with theparent strain, and low similarity with the expected transformedsequences. Data points towards the top left of the chart exhibit highsimilarity to expected transformed sequences and low identity withparent strain. Data points in the middle likely represent heterokaryonswith multiple nuclei.

FIG. 27 depicts a large scale protoplasting method in which multiplebatches of 500 ml cultures are subjected to protoplasting in 500 ml ofprotoplasting buffer followed by storing the generated protoplasts at−80 C. This is method is scaled up as compared to using 100 ml culturesin 50-100 ml protoplasting buffer.

FIG. 28 depicts a protoplasting portion of a workflow for ahigh-throughput (HTP) system for building strains of coenocyticorganisms (e.g., filamentous fungi)

FIG. 29 depicts the minimum inhibitory concentration (MIC) of thechemical inhibitor W-7 on two strains (1015; 11414) of Aspergillus niger(A. niger).

FIG. 30 depicts steps to rapidly isolate genomic DNA and prepareamplicons that contain identifying sequences that associate specificamplicons with the well that they came from which contains the organismthat was isolated following genetic alteration. The method allows forisolation and screening of 96 transformants/transformations.Transformants are plated to Omnitrays, allowed to sporulate and asterile 96 pin replicator used to rapidly isolate.

FIG. 31 depicts picking of transformants and their subsequent transferto and sporulation in 96-well microtiter seed plates. The colonies arepicked with toothpicks (K-picks). The seed plate contains 400 μl agarmedia. Spore suspensions made from the seed plate are used to make Sporeplate, and stamping/screening are done from Spore plate.

FIG. 32 depicts a fragment analyzer run following amplification ofnucleic acid fragments following extraction of DNA from fungaltransformants using a boil prep method as provided herein. Preparinggenomic template from multiple samples in fungi can be challenging sincethe cell wall makes grinding often necessary, obtaining DNA from sporescan be difficult and spore PCR does not work well with most fungi. Asshown, short length PCRs for NGS was performed successfully. The boilprep method showed that PrepMan can be used as an effective method forgenomic DNA purification, and can be automated.

FIG. 33 depicts colorimetric differences in strains containing a mixtureof spores of difference genetic background (i.e., mutant (m) vs.parental (p)) at different ratios. As shown, only strains with noparental spores (0:1 p/m ratio) appear yellow in the tested selectionscheme. There two ways to determine if a transformant is a heterokaryon:1.) phenotype and 2.) Sequencing. In this figure, the transformants werescored by yellow phenotype and NGS. Testing the sensitivity of these twomethods is important for adapting transformant scoring to a workflow. If1/10 of the nucleic are black, the colony may appear black and thus NGScan be used before and after counterselection.

FIG. 34 depicts a plot of the amplicon sequences from the mixed sporeplate shown in FIG. 33 and the plate at the top of FIG. 35. Thepercentage of amplicons that contained the targeted mutation is on the Yaxis and the percentage that still contain the parent is on the X axis.The large plot contains data from the entire plate demonstrating acomplete range of mixed spores within the plate. The graphs on the rightare of the individual rows from the plate. These graphs of the rowsdefine the range of amplicon distribution that is observed in NGS when adefined mixture of nuclei are tested. This analyses can then be used topredict the distribution of SNPs within a data set from a SNPSWP strainbuild. This prediction can be used to QC steps in a strain buildprocess.

FIG. 35 depicts a test of the ability of NGS to detect SNPS in a mixtureof spores from filamentous fungi with different genetic backgrounds thatgive rise to strains with either a pyrG-background (black) ormet3-background (light colored). The met3-strains require growth mediawith methionine and are resistant to selenite. The plate containingspores at the top demonstrates that phenotypes can be masked in mixedcultures and can be difficult to score visually. The row that contains10 fold more mutant (yellow) nuclei still appears black. NGS can detectthe mutation where visual phenotype cannot. The pie charts are anexample of how the population can shift under different growthconditions. In particular, NGS showed that selective media can forcemixed populations of nuclei to homokaryon. This can be utilized forstrain purification but it also demonstrates that growth and propagationduring the process of strain building must be monitored at theindividual SNP level.

FIG. 36 depicts three approaches (Spilt marker with SNP repeats; SpiltMarker Terminator Repeats; Loop-in Single Crossover) for performing SNPintegration. The split marker with SNP repeats can be used to generatean insertional null as described in the present disclosure. The splitmarker terminator repeats has the advantage that it may facilitate thedesired phenotype from a primary integrant. In contrast, the loop-insingle crossover requires plasmid cloning cloning and preparation,ectopic integration may be high and Concatemers may occur.

FIG. 37 depicts an embodiment utilizing bipartite marker transformationfor performing combinatorial SNPSWP in a coenocytic organism such as afilamentous fungi. This figure depicts a tool for combinatorial SNPSWPin fungi that can combine various inducible promoters with divergentpyrG genes and promoters that can be catabolite repressed by glucose,Transformants can be selected on glucose such that multiple integrationsof weakly expressed genes will be favored and transformants can beplated on induction media with FOA to get loop-outs.

FIGS. 38A-C depicts use of NGS sequencing to detect multipleintegrations, ectopic integration or the presence of SNPs and non-SNPsin the same nuclei.

FIG. 39 depicts performing SNPSWP by generating gene deletions.

FIG. 40 depicts four different promoters being placed in front of atarget gene to generate 4 different strains. These strains can the becompared in a test for a desired trait and an ideal level of expressioncan be determined.

FIG. 41 illustrates an example of the distribution of relative strainperformances for the input data under consideration done inCorynebacterium by using the method described in the present disclosure.A relative performance of zero indicates that the engineered strainperformed equally well to the in-plate base strain. The processesdescribed herein are designed to identify the strains that are likely toperform significantly above zero.

FIG. 42 illustrates the use of SNP SWP to integrate two BP changes asdemonstrated by restriction analysis of amplicons. The results depictedin this figure are for an experiment whereby a SNPSWP was performed in akusA+ strain. Here, an EcoRV restriction site was targeted via SNPSWP inorder to add two bp SNP that change the EcoRV site to a BamHIrestriction site (see SEQ ID NO: 20). The pyrG gene was targeted to theaygA locus in order to allow for colorimetric selection (i.e., pickyellow colonies) and amplification was followed by restriction digestionto screen for integration of the SNP. Of the 36 yellow transformantspicked, 24/36 contained a BamHI site in the amplicon. Thus, SNPSWPco-transformation works without kusA.

FIG. 43 illustrates how a strain and its improved descendants differ intheir response to Citric Acid production media.

FIG. 44 illustrates an empirical design strategy to systematically andcomprehensively explore the genome independent of defined genefunctions. Depicted in this figure are 2 strains that differ in theirgenome by 43 SNPs. Using the SNPSWP methods provided throughout thepresent disclosure, the role of each of these SNP alone or incombination can be examined with automation.

FIG. 45 illustrates the use of fusion PCR the generate split-markerconstructs for use in the present invention.

FIG. 46A-B illustrates design and generation (see FIG. 46A) as well asquality control analysis using a fragment analyzer (see FIG. 46B) ofsplit-marker constructs generated as depicted in FIG. 45.

FIG. 47 illustrates the annealing of crRNA to tracrRNA and complexed toCas9 protein, thereby creating an RNP capable of crRNA-directed DNAcleavage.

FIG. 48 illustrates the method for transforming RNP into Aspergillusniger protoplasts. 100 uL or 10{circumflex over ( )}6 protoplasts(PyrG−) are transformed with RNP and a plasmid containing a PyrG markerin 10 ul of STC buffer. These are mixed and incubated on ice for fifteenminutes. The cells are then mixed with 40% PEG in STC and placed at roomtemperature for 15 minutes. Transformants are mixed in osmoticallystabilized minimal media with +0.8% agarose and overlayed withadditional agar. Colonies are then counted and scored for changes inphenotype caused by RNP-mediated genome editing (not shown).

FIG. 49A-49F illustrates the non-homologous end joining (NHEJ) repair ofthe Cas9 RNP cleavage at the AygA locus. The AygA gene is targeted byone or two crRNA sequences complexed to tracrRNA and Cas9 (FIG. 49A).Indels result in a change in conidia color from (FIG. 49B) black to(FIG. 49C) yellow, enabling a phenotypic screen for successful RNPtransformation. An example of a trace file from amplified genomic DNAisolated from protoplasts transformed with a single crRNA complexed to atracrRNA and Cas9 protein demonstrates that an indel has formed proximalto the target site (FIG. 49D; see SEQ ID NOs 21 and 22). A trace file ofamplified genomic DNA isolated from transformations with RNPs targetingtwo sites 771 bp apart suggests that both RNPs can co-transform into asingle protoplast and mediate a large internal deletion between twotarget sites (FIG. 49E; see SEQ ID NO: 23). Number of colony formingunits (CFUs) of a transformation experiment and the estimated percent ofthose colonies containing indels when transformed with 1, 2 or controlcrRNA/tracrRNA sequences (FIG. 49F). The CFUs are counted after a 10×dilution of the total transformation.

FIG. 50A-50C illustrates the measurement of the efficiency of HRmediated by linear donors and an RNP targeting the genome. Protoplastsare co-transformed Cas9 complexed to one (Ayg.1) or two (Ayg.1+Ayg.3)cr/tracrRNAs targeting the AygA gene as well as a linear donor and aplasmid containing pyrG. The donor is flanked with 487 or 438 bp ofhomology around the Ayg.1 cut site. The donor contains (FIG. 50A) a pyrGgene with a promoter and terminator or (FIG. 50B) a 4 bp insertion. TheAygA locus was PCR amplified from single germinated spores (FIG. 50C).Results demonstrate that contransforming RNPs, plasmid and a donormediates insertion of the pyrG gene in the presence of targeted crRNAbut not control crRNAs. This experiment also shows that an RNPco-transformation with donor from (FIG. 50B) enables an 86% HR editingrate.

FIG. 51 illustrates the DJV_03_pyrG_insertion_in_AygA shows pyrG withpromoter and terminator (lowercase) flanked by 5′ and 3′ regions ofhomology (uppercase) to the AygA gene. This figure corresponds to SEQ IDNO:9.

FIG. 52 illustrates the DJV_07_4 bp_insertion_in_AygA contains a 4 bpinsertion (lowercase) flanked by 5′ and 3′ regions of homology(uppercase) to the AygA gene. This figure corresponds to SEQ ID NO:10.

FIG. 53 illustrates traditional strategies for introducing or changingsequences in a genome. A split gene marker (left) or integrationconstruct (right) can be used to incorporate new genetic material viathree or one cross-over event(s) respectively. Regions of homology,surrounding the marker and mutation target the integration to a desiredlocus. Later, the marker can be used to select for the loop-in event anda counterselectable marker can then be used to select for the loop-outevent. The integrants may loop out producing the wild type sequenceshown in (1) or the new mutation shown in (3).

FIG. 54A-54B illustrate a workflows for modifying Aspergillus nigerutilizing traditional and new methods. The traditional workflow takes 20days and clonal populations are not explicitly achieved (FIG. 54A).Growth on minimal media inhibits parental strain from growing, but doesnot inhibit heterokaryons, which contain both transformed anduntransformed nuclei in the same cell. The new protocol results inparent death and pure-clonal populations at step 2 and takes only 12days (FIG. 54B).

FIG. 55A, FIG. 55B, and FIG. 55C depict one print single spores withhigh fidelity, dispensed by the CellenONE (Cellenion, Lyon, FR). Yellowand black spores were mixed 1:1 in water at a final concentration of2×10⁶ and dispensed by the CellenONE (Cellenion, Lyon, FR) into three×96well and one×384 well microtiter plates containing agar. After 4 days,wells were visually counted (FIG. 55A). Image of a 96 well plate printedwith back and yellow spores (FIG. 55B). Percentage of wells that did notcontain a germinating spore. This could be due to a misprint (dispensingnothing) or to printing of a non-viable spore (FIG. 55C). Percentage ofwells showing both black and yellow spores, indicating that two sporeswere printed in the same droplet.

FIG. 56A-B illustrate that two annealed oligos can create a SNP withoutaltering the PAM or seed region of the protospacer site. A doublestranded donor was cotransformed with RNP and a plasmid as described inExample 11. FIG. 56A shows the donor contains a nonsense mutation (shownin lower case in SEQ ID NO: 24) flanked on the 5′ and 3′ sides by 50 bphomology to the AygA gene. FIG. 56B shows two trace files of isolatedcolonies sequenced with Sanger technology. The first aligns to thewildtype sequence (see SEQ ID NO: 25) and the second trace file containsthe intended mutation (indicated by an asterisk; see SEQ ID NO: 26).

FIG. 57 illustrates promoter swapping of morphology gene (i.e.,FungiSNP_18; SEQ ID NO: 13). Different promoters controlling expressionof this gene impact morphology. The strains containing the manB fusionand the amyB fusion retain the multiple tips vs. the 11414 parentstrain, whereas those with higher expression srpB and mbfA lack themultiple tip phenotype. The strains were grown in citric acid productionmedia (14% w/v Glucose, pH 2, depleted Mn++) at 30° C. for 48 hours.When allowed to incubate for 168 hours, the strains with higherexpression promoters as well as the parent control all contained longfilamentous hyphae. The strains with the lower level of expression fromthe promoter fusion, amyB and manB, remained pelleted.

FIG. 58 illustrates promoter swapping of morphology gene target 18 inthe base 1015 strain and 11414 production strain. The gene productassociated with FungiSNP_18 is a signaling kinase that responds toosmotic stress (i.e., A. niger ortholog of S. cerevisiae SLN1). Thisfigure shows that when the gene expression of said gene is reduced byreplacing the native promoter with a weaker promoter, the cells maintaina tighter, less elongated phenotype, which is referred to herein as a‘pellet’ phenotype (see right hand panels for the cells expressing themanB(p)snp18 gene in the base 1015 strain and 11414 production strain).The strains were grown in citric acid production media (14% w/v Glucose,pH 2, depleted Mn++) at 30° C. for 24 hours. This type of growth can befavorable to stirred tank fermentation.

FIG. 59 illustrates that reduced levels of the FungiSNP_18 gene productin the base strain (i.e., A. niger 1015) by introducing the FungiSNP_18gene (SEQ ID NO: 13) under the control of the manB(p) promoter (SEQ IDNO: 1) results in inability to sporulate in the base strain geneticbackground. This phenotype was not observed when the same construct wasintroduced to the production strain (i.e., A. niger 11414).

FIG. 60 illustrates the results of A. niger split marker designtransformation and validation according to the methods of the presentdisclosure. The data was generated using NGS of transformed (via splitmarker) A. niger mutants and is a distribution of the match to themutation at the target vs. match to parent at the target. Every samplein the top left corner of this graph are correct and have passed QC. Thesamples within the circle contain both the mutant and parent at thelocus and may be processed again through steps 4 and 5 of FIG. 20B inorder to generate isolates that may pass QC.

FIG. 61 is a graphic representation of the NGS data from a SNPSWPcampaign. In this example, 31 loci were targeted using constructsdesigned as presented in FIG. 45. Here 1264 total isolates were screenedby sequencing each amplicon populations from all individual samples.This data set contained over one million sequenced amplicons. There were119 samples that passed all QC requirements. Quality control includeschecking for the presence of parental mutation at the loci and all ofthe amplicons from the well must match the target DNA across the entireamplicon. Samples with the + symbol are correct, samples that have thedot symbol may contain both the parent and the mutation.

FIG. 62 illustrates that strains that contain the Base SNP18 grow fasteron low pH media.

FIG. 63 illustrates that strains that contain the Base SNP18 grow fasteron media which provide osmotic stress.

FIG. 64 illustrates that exchanging FungiSNP_18 between the base andproduction strains has an impact on sporulation and radial growth rate.

FIG. 65 illustrates deletion in the base strain of all coding sequencesthat contain SNPs (i.e., the FungiSNPs from Table 4) in the productionstrain.

FIG. 66 illustrates that the gene that contains FungiSNP18 isdispensible for sporulation in the production strain but not in the basestrain.

FIG. 67 illustrates the design of the bipartite constructs and generalscheme employed for conducting the PROSWP experiments described inExample 3.

FIG. 68 illustrates that weaker promoters used in Example 3 impactmorphology. The strain containing FungiSNP_18 (SNP18) under the weakmanB promoter has tighter colony morphology than strains containingother promoter combinations. The impact of SNP18 control is morepronounced under osmotic stress than under low pH.

FIG. 69 illustrates the PROSWP of FungiSNP_12 (snp_12), Lower strengthpromoters operably linked to snp_12 and result in yellow pigment inhyphae and some altered morphology (observed at the edge of colonies).This yellow pigment is common in a variety of mutants and is thought ofas a sign of metabolic stress.

FIG. 70 illustrates that when driven by weaker promoters, FungiSNP_18(snp_18) has more severe morphological phenotype in the base strain thanin the production strain.

DETAILED DESCRIPTION

The current disclosure overcomes many of the challenges inherent ingenetically manipulating filamentous fungi in an automated,high-throughput platform. The methods provided herein are designed togenerate fungal production strains by incorporating genetic changesusing automated co-transformation combined with automated screening oftransformants thereby allowing exchange of genetic traits between twostrains without going through a sexual cross. This disclosure alsoincludes a procedure for generating large numbers of protoplasts and ameans to store them for later use. Large batches of readily availablecompetent cells can greatly facilitate automation.

Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” refers to one or more of that entity, i.e. canrefer to a plural referents. As such, the terms “a” or “an”, “one ormore” and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

As used herein the terms “cellular organism” “microorganism” or“microbe” should be taken broadly. These terms are used interchangeablyand include, but are not limited to, the two prokaryotic domains,Bacteria and Archaea, as well as certain eukaryotic fungi (e.g.,filamentous fungi described herein) and protists. In some embodiments,the disclosure refers to the “microorganisms” or “cellular organisms” or“microbes” of lists/tables and figures present in the disclosure. Thischaracterization can refer to not only the identified taxonomic generaof the tables and figures, but also the identified taxonomic species, aswell as the various novel and newly identified or designed strains ofany organism in said tables or figures. The same characterization holdstrue for the recitation of these terms in other parts of theSpecification, such as in the Examples.

The term “coenocyte” or “coenocytic organism” as used herein can referto a multinucleate cell or an organism comprising a multinucleate cell.The multinucleate cell can result from multiple nuclear divisionswithout their accompanying cytokinesis, in contrast to a syncytium,which results from cellular aggregation followed by dissolution of thecell membranes inside the mass. Examples of coenocytic organisms as itpertains to the methods, compositions and systems provided herein caninclude protists (e.g., algae, protozoa, myxogastrids (slime molds),alveolates, plants, fungi (e.g., filamentous fungi), and/or metazoans(e.g., Drosophila spp).

The term “prokaryotes” is art recognized and refers to cells whichcontain no nucleus or other cell organelles. The prokaryotes aregenerally classified in one of two domains, the Bacteria and theArchaea. The definitive difference between organisms of the Archaea andBacteria domains is based on fundamental differences in the nucleotidebase sequence in the 16S ribosomal RNA.

The term “Archaea” refers to a categorization of organisms of thedivision Mendosicutes, typically found in unusual environments anddistinguished from the rest of the prokaryotes by several criteria,including the number of ribosomal proteins and the lack of muramic acidin cell walls. On the basis of ssrRNA analysis, the Archaea consist oftwo phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota.On the basis of their physiology, the Archaea can be organized intothree types: methanogens (prokaryotes that produce methane); extremehalophiles (prokaryotes that live at very high concentrations of salt(NaCl); and extreme (hyper) thermophilus (prokaryotes that live at veryhigh temperatures). Besides the unifying archaeal features thatdistinguish them from Bacteria (i.e., no murein in cell wall,ester-linked membrane lipids, etc.), these prokaryotes exhibit uniquestructural or biochemical attributes which adapt them to theirparticular habitats. The Crenarchaeota consists mainly ofhyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeotacontains the methanogens and extreme halophiles.

“Bacteria” or “eubacteria” refers to a domain of prokaryotic organisms.Bacteria include at least 11 distinct groups as follows: (1)Gram-positive (gram+) bacteria, of which there are two majorsubdivisions: (1) high G+C group (Actinomycetes, Mycobacteria,Micrococcus, others) (2) low G+C group (Bacillus, Clostridia,Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

A “eukaryote” is any organism whose cells contain a nucleus and otherorganelles enclosed within membranes. Eukaryotes belong to the taxonEukarya or Eukaryota. The defining feature that sets eukaryotic cellsapart from prokaryotic cells (the aforementioned Bacteria and Archaea)is that they have membrane-bound organelles, especially the nucleus,which contains the genetic material, and is enclosed by the nuclearenvelope.

The terms “genetically modified host cell,” “recombinant host cell,” and“recombinant strain” are used interchangeably herein and refer to hostcells that have been genetically modified by the cloning andtransformation methods of the present disclosure. Thus, the termsinclude a host cell (e.g., bacteria, yeast cell, fungal cell, CHO, humancell, etc.) that has been genetically altered, modified, or engineered,such that it exhibits an altered, modified, or different genotype and/orphenotype (e.g., when the genetic modification affects coding nucleicacid sequences of the microorganism), as compared to thenaturally-occurring organism from which it was derived. It is understoodthat in some embodiments, the terms refer not only to the particularrecombinant host cell in question, but also to the progeny or potentialprogeny of such a host cell.

The term “wild-type microorganism” or “wild-type host cell” describes acell that occurs in nature, i.e. a cell that has not been geneticallymodified.

The term “parent strain” or “parental strain” or “parent” may refer to ahost cell from which mutant strains are derived. Accordingly, the“parent strain” or “parental strain” is a host cell or cell whose genomeis perturbed by any manner known in the art and/or provided herein togenerate one or more mutant strains. The “parent strain” or “parentalstrain” may or may not have a genome identical to that of a wild-typestrain.

The term “genetically engineered” may refer to any manipulation of ahost cell's genome (e.g. by insertion, deletion, mutation, orreplacement of nucleic acids).

The term “control” or “control host cell” refers to an appropriatecomparator host cell for determining the effect of a geneticmodification or experimental treatment. In some embodiments, the controlhost cell is a wild type cell. In other embodiments, a control host cellis genetically identical to the genetically modified host cell, save forthe genetic modification(s) differentiating the treatment host cell. Insome embodiments, the present disclosure teaches the use of parentstrains as control host cells (e.g., the S₁ strain that was used as thebasis for the strain improvement program). In other embodiments, a hostcell may be a genetically identical cell that lacks a specific promoteror SNP being tested in the treatment host cell.

As used herein, the term “allele(s)” means any of one or morealternative forms of a gene, all of which alleles relate to at least onetrait or characteristic. In a diploid cell, the two alleles of a givengene occupy corresponding loci on a pair of homologous chromosomes.Since the present disclosure, in embodiments, relates to QTLs, i.e.genomic regions that may comprise one or more genes or regulatorysequences, it is in some instances more accurate to refer to “haplotype”(i.e. an allele of a chromosomal segment) instead of “allele”, however,in those instances, the term “allele” should be understood to comprisethe term “haplotype”.

As used herein, the term “locus” (loci plural) means a specific place orplaces or a site on a chromosome where for example a gene or geneticmarker is found.

As used herein, the term “genetically linked” refers to two or moretraits that are co-inherited at a high rate during breeding such thatthey are difficult to separate through crossing.

A “recombination” or “recombination event” as used herein refers to achromosomal crossing over or independent assortment. The term“recombinant” refers to an organism having a new genetic makeup arisingas a result of a recombination event.

As used herein, the term “phenotype” refers to the observablecharacteristics of an individual cell, cell culture, organism, or groupof organisms which results from the interaction between thatindividual's genetic makeup (i.e., genotype) and the environment.

As used herein, the term “chimeric” or “recombinant” when describing anucleic acid sequence or a protein sequence refers to a nucleic acid, ora protein sequence, that links at least two heterologouspolynucleotides, or two heterologous polypeptides, into a singlemacromolecule, or that re-arranges one or more elements of at least onenatural nucleic acid or protein sequence. For example, the term“recombinant” can refer to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

As used herein, a “synthetic nucleotide sequence” or “syntheticpolynucleotide sequence” is a nucleotide sequence that is not known tooccur in nature or that is not naturally occurring. Generally, such asynthetic nucleotide sequence will comprise at least one nucleotidedifference when compared to any other naturally occurring nucleotidesequence.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “DNA scaffold” or “nucleic acid scaffold”refers to a nucleic acid scaffold that is either artificially producedor a naturally occurring sequence that is repurposed as a scaffold. Inone embodiment of the present disclosure, the nucleic acid scaffold is asynthetic deoxyribonucleic acid scaffold. The deoxyribonucleotides ofthe synthetic scaffold may comprise purine and pyrimidine bases or othernatural, chemically or biochemically modified, non-natural, orderivatized deoxyribonucleotide bases. As described in more detailherein, the nucleic acid scaffold of the present disclosure is utilizedto spatially and temporally assemble and immobilize two or more proteinsinvolved in a biological pathway, i.e. biosynthetic enzymes, to create afunctional complex. The assembly and immobilization of each biologicalpathway protein on the scaffold occurs via the binding interactionbetween one of the protein-binding sequences, i.e., protein dockingsites, of the scaffold and a corresponding DNA-binding portion of achimeric biosynthetic enzyme. Accordingly, the nucleic acid scaffoldcomprises one or more subunits, each subunit comprising two or moreprotein-binding sequences to accommodate the binding of two or moredifferent chimeric biological pathway proteins.

As used herein, a “DNA binding sequence” or “DNA binding site” refers toa specific nucleic acid sequence that is recognized and bound by aDNA-binding domain portion of a chimeric biosynthetic genes of thepresent disclosure. Many DNA-binding protein domains and their cognatebinding partner recognition sites (i.e., protein binding sites) are wellknown in the art. For example, numerous zinc finger binding domains andtheir corresponding DNA protein binding target sites are known in theart and suitable for use in the present disclosure. Other DNA bindingdomains include, without limitation, leucine zipper binding domains andtheir corresponding DNA protein binding sites, winged helix bindingdomains and their corresponding DNA protein binding sites, wingedhelix-turn-helix binding domains and their corresponding DNA proteinbinding sites, HMG-box binding domains and their corresponding DNAprotein binding sequences, helix-loop-helix binding domains and theircorresponding DNA protein binding sequences, and helix-turn-helixbinding domains and their corresponding DNA protein binding sequences.Other known DNA binding domains with known DNA protein binding sequencesinclude the immunoglobulin DNA domain, B3 DNA binding domain, and TALeffector DNA binding domain. Nucleic acid scaffold subunits of thepresent disclosure may comprises any two or more of the aforementionedprotein binding sites.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include non-expressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “homologous” or “homologue” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and are determined based on the degree ofsequence identity. The terms “homology,” “homologous,” “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein. They refer to nucleic acid fragments wherein changes in one ormore nucleotide bases do not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant disclosure such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the disclosure encompasses more than the specificexemplary sequences. These terms describe the relationship between agene found in one species, subspecies, variety, cultivar or strain andthe corresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this disclosure homologoussequences are compared. “Homologous sequences” or “homologues” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus(Scientific and Educational Software, Pennsylvania) and AlignX (VectorNTI, Invitrogen, Carlsbad, Calif.). Another alignment program isSequencher (Gene Codes, Ann Arbor, Mich.), using default parameters.

As used herein, the term “endogenous” or “endogenous gene,” refers tothe naturally occurring gene, in the location in which it is naturallyfound within the host cell genome. In the context of the presentdisclosure, operably linking a heterologous promoter to an endogenousgene means genetically inserting a heterologous promoter sequence infront of an existing gene, in the location where that gene is naturallypresent. An endogenous gene as described herein can include alleles ofnaturally occurring genes that have been mutated according to any of themethods of the present disclosure.

As used herein, the term “exogenous” is used interchangeably with theterm “heterologous,” and refers to a substance coming from some sourceother than its native source. For example, the terms “exogenousprotein,” or “exogenous gene” refer to a protein or gene from anon-native source or location, and that have been artificially suppliedto a biological system.

As used herein, the term “heterologous modification” can refer to amodification coming from a source other than a source native to aparticular biological system (e.g., a host cell as provided herein), ora modification from a source that is native to the particular biologicalsystem, but which is found in a non-native context/position/location.Thus, the modification is non-native or not naturally occurring inreference to a biological system (e.g., a host cell as provided herein,or non-native context/position/location within a host cell), in whichsaid modification has been or will be introduced. The heterologousmodification can therefore be considered artificially introduced to thebiological system (e.g., a host cell as provided herein, or heterologouscontext/position/location within a host). The modification can be agenetic or epigenetic variation, disruption or perturbation. A geneticvariation, disruption or perturbation can be, for example, replacementof a native promoter and/or terminator of a gene with a promoter and/orterminator that is not native to said host, or it can be a promoterand/or terminator from within the host organism that has been moved to anon-native heterologous context/position/location. A genetic variation,disruption or perturbation can be replacement of a native or naturallyoccurring gene with a non-native or naturally occurring gene such as,for example a selectable marker gene. Or, a genetic variation,disruption or perturbation can be replacement, or swapping, of a nativeor naturally occurring gene, with another native gene (e.g. promoter)from within the host genome, which is placed into a non-naturalcontext/position/location. A genetic variation, disruption orperturbation can be replacement of a native or naturally occurring genewith a non-native or naturally occurring form of the gene. Thenon-native or naturally occurring form of the gene can be a mutant formof the gene not naturally found in a particular host cell and/or amutant form of the gene not naturally found in a particular host celloperably linked to a heterologous promoter and/or terminator.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids.

Variant polynucleotides also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.Biol. 272:336-347; Zhang el al. (1997) PNAS 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (2001) Molecular Cloning: A Laboratory Manual (3^(rd) ed., ColdSpring Harbor Laboratory Press, Plainview, N.Y.). See also Innis el al.,eds. (1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The terms “stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimized to maximize specificbinding and minimize non-specific binding of primer or probe to itstarget nucleic acid sequence. The terms as used include reference toconditions under which a probe or primer will hybridize to its targetsequence, to a detectably greater degree than other sequences (e.g. atleast 2-fold over background). Stringent conditions are sequencedependent and will be different in different circumstances. Longersequences hybridize specifically at higher temperatures. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe or primer. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M Na+ion, typically about 0.01 to 1.0 M Na+ ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes or primers (e.g. 10 to 50 nucleotides) and at least about60° C. for long probes or primers (e.g. greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. Exemplary low stringentconditions or “conditions of reduced stringency” include hybridizationwith a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. anda wash in 2×SSC at 40° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in0.1×SSC at 60° C. Hybridization procedures are well known in the art andare described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001. Insome embodiments, stringent conditions are hybridization in 0.25 MNa2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5-20% sodium dodecylsulfate at 45° C., such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by awash in 5×SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55° C.to 65° C.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Insome embodiments, the promoter sequence consists of proximal and moredistal upstream elements, the latter elements often referred to asenhancers. Accordingly, an “enhancer” is a DNA sequence that canstimulate promoter activity, and may be an innate element of thepromoter or a heterologous element inserted to enhance the level ortissue specificity of a promoter. Promoters may be derived in theirentirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. A promoter for usein the methods and systems described herein can be inducible such thatexpression of a gene or genes under control of said promoter isregulated by the presence and/or absence of a specific agent. Theinducible promoters can be any promoter whose transcriptional activityis regulated by the presence or absence of a chemical or a physicalcondition such as for example, alcohol, tetracycline, steroids, metal orother compounds known in the art or by the presence or absence of lightor low or high temperatures. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity.

As used herein, “terminator” generally refers to a section of DNAsequence that marks the end of a gene in genomic DNA and is capable ofstopping transcription. Terminators may be derived in their entiretyfrom a native gene, or be composed of different elements derived fromdifferent terminators found in nature, or even comprise synthetic DNAsegments. It is understood by those skilled in the art that differentterminators may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the disclosure. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. Vectors can be plasmids, viruses,bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. As used herein, the term“expression” refers to the production of a functional end-product e.g.,an mRNA or a protein (precursor or mature).

“Operably linked” means in this context the sequential arrangement ofthe promoter polynucleotide according to the disclosure with a furtheroligo- or polynucleotide, resulting in transcription of said furtherpolynucleotide.

The term “product of interest” or “biomolecule” as used herein refers toany product produced by microbes from feedstock. In some cases, theproduct of interest may be a small molecule, enzyme, peptide, aminoacid, organic acid, synthetic compound, fuel, alcohol, etc. For example,the product of interest or biomolecule may be any primary or secondaryextracellular metabolite. The primary metabolite may be, inter alia,ethanol, citric acid, lactic acid, glutamic acid, glutamate, lysine,threonine, tryptophan and other amino acids, vitamins, polysaccharides,etc. The secondary metabolite may be, inter alia, an antibiotic compoundlike penicillin, or an immunosuppressant like cyclosporin A, a planthormone like gibberellin, a statin drug like lovastatin, a fungicidelike griseofulvin, etc. The product of interest or biomolecule may alsobe any intracellular component produced by a microbe, such as: amicrobial enzyme, including: catalase, amylase, protease, pectinase,glucose isomerase, cellulase, hemicellulase, lipase, lactase,streptokinase, and many others. The intracellular component may alsoinclude recombinant proteins, such as: insulin, hepatitis B vaccine,interferon, granulocyte colony-stimulating factor, streptokinase andothers. The product of interest may also refer to a “protein ofinterest”.

The term “protein of interest” generally refers to any polypeptide thatis desired to be expressed in a filamentous fungus. Such a protein canbe an enzyme, a substrate-binding protein, a surface-active protein, astructural protein, or the like, and can be expressed at high levels,and can be for the purpose of commercialization. The protein of interestcan be encoded by an endogenous gene or a heterologous gene relative tothe variant strain and/or the parental strain. The protein of interestcan be expressed intracellularly or as a secreted protein. If theprotein of interest is not naturally secreted, the polynucleotideencoding the protein may be modified to have a signal sequence inaccordance with techniques known in the art. The proteins, which aresecreted may be endogenous proteins which are expressed naturally, butcan also be heterologous. Heterologous means that the gene encoded bythe protein is not produced under native condition in the filamentousfungal host cell. Examples of enzymes which may be produced by thefilamentous fungi of the disclosure are carbohydrases, e.g. cellulasessuch as endoglucanases, beta-glucanases, cellobiohydrolases orbeta-glucosidases, hemicellulases or pectinolytic enzymes such asxylanases, xylosidases, mannanases, galactanases, galactosidases,rhamnogalacturonases, arabanases, galacturonases, lyases, or amylolyticenzymes; phosphatases such as phytases, esterases such as lipases,proteolytic enzymes, oxidoreductases such as oxidases, transferases, orisomerases.

The term “carbon source” generally refers to a substance suitable to beused as a source of carbon for cell growth. Carbon sources include, butare not limited to, biomass hydrolysates, starch, sucrose, cellulose,hemicellulose, xylose, and lignin, as well as monomeric components ofthese substrates. Carbon sources can comprise various organic compoundsin various forms, including, but not limited to polymers, carbohydrates,acids, alcohols, aldehydes, ketones, amino acids, peptides, etc. Theseinclude, for example, various monosaccharides such as glucose, dextrose(D-glucose), maltose, oligosaccharides, polysaccharides, saturated orunsaturated fatty acids, succinate, lactate, acetate, ethanol, etc., ormixtures thereof. Photosynthetic organisms can additionally produce acarbon source as a product of photosynthesis. In some embodiments,carbon sources may be selected from biomass hydrolysates and glucose.

The term “feedstock” is defined as a raw material or mixture of rawmaterials supplied to a microorganism or fermentation process from whichother products can be made. For example, a carbon source, such asbiomass or the carbon compounds derived from biomass are a feedstock fora microorganism that produces a product of interest (e.g. smallmolecule, peptide, synthetic compound, fuel, alcohol, etc.) in afermentation process. However, a feedstock may contain nutrients otherthan a carbon source.

The term “volumetric productivity” or “production rate” is defined asthe amount of product formed per volume of medium per unit of time.Volumetric productivity can be reported in gram per liter per hour(g/L/h).

The term “specific productivity” is defined as the rate of formation ofthe product. Specific productivity is herein further defined as thespecific productivity in gram product per gram of cell dry weight (CDW)per hour (g/g CDW/h). Using the relation of CDW to OD₆₀₀ for the givenmicroorganism specific productivity can also be expressed as gramproduct per liter culture medium per optical density of the culturebroth at 600 nm (OD) per hour (g/L/h/OD).

The term “yield” is defined as the amount of product obtained per unitweight of raw material and may be expressed as g product per g substrate(g/g). Yield may be expressed as a percentage of the theoretical yield.“Theoretical yield” is defined as the maximum amount of product that canbe generated per a given amount of substrate as dictated by thestoichiometry of the metabolic pathway used to make the product.

The term “titre” or “titer” is defined as the strength of a solution orthe concentration of a substance in solution. For example, the titre ofa product of interest (e.g. small molecule, peptide, synthetic compound,fuel, alcohol, etc.) in a fermentation broth is described as g ofproduct of interest in solution per liter of fermentation broth (g/L).

The term “total titer” is defined as the sum of all product of interestproduced in a process, including but not limited to the product ofinterest in solution, the product of interest in gas phase ifapplicable, and any product of interest removed from the process andrecovered relative to the initial volume in the process or the operatingvolume in the process

As used herein, the term “HTP genetic design library” or “library”refers to collections of genetic perturbations according to the presentdisclosure. In some embodiments, the libraries of the present disclosuremay manifest as i) a collection of sequence information in a database orother computer file, ii) a collection of genetic constructs encoding forthe aforementioned series of genetic elements, or iii) host cell strainscomprising said genetic elements. In some embodiments, the libraries ofthe present disclosure may refer to collections of individual elements(e.g., collections of promoters for PRO swap libraries, or collectionsof terminators for STOP swap libraries). In other embodiments, thelibraries of the present disclosure may also refer to combinations ofgenetic elements, such as combinations of promoter::genes,gene:terminator, or even promoter:gene:terminators. In some embodiments,the libraries of the present disclosure further comprise meta dataassociated with the effects of applying each member of the library inhost organisms. For example, a library as used herein can include acollection of promoter::gene sequence combinations, together with theresulting effect of those combinations on one or more phenotypes in aparticular species, thus improving the future predictive value of usingsaid combination in future promoter swaps.

As used herein, the term “SNP” can refer to Small NuclearPolymorphism(s). In some embodiments, SNPs of the present disclosureshould be construed broadly, and include single nucleotidepolymorphisms, sequence insertions, deletions, inversions, and othersequence replacements. As used herein, the term “non-synonymous” ornon-synonymous SNPs” can refer to mutations that lead to coding changesin host cell proteins

A “high-throughput (HTP)” method of genomic engineering may involve theutilization of at least one piece of automated equipment (e.g. a liquidhandler or plate handler machine) to carry out at least one step of saidmethod.

The CRISPR/Cas system is a prokaryotic immune system that confersresistance to foreign genetic elements such as those present withinplasmids and phages and that provides a form of acquired immunity.CRISPR stands for Clustered Regularly Interspaced Short PalindromicRepeat, and cas stands for CRISPR-associated system, and refers to thesmall cas genes associated with the CRISPR complex.

CRISPR-Cas systems are most broadly characterized as either Class 1 orClass 2 systems. The main distinguishing feature between these twosystems is the nature of the Cas-effector module. Class 1 systemsrequire assembly of multiple Cas proteins in a complex (referred to as a“Cascade complex”) to mediate interference, while Class 2 systems use alarge single Cas enzyme to mediate interference. Each of the Class 1 andClass 2 systems are further divided into multiple CRISPR-Cas types basedon the presence of a specific Cas protein. For example, the Class 1system is divided into the following three types: Type I systems, whichcontain the Cas3 protein; Type III systems, which contain the Cas10protein; and the putative Type IV systems, which contain the Csf1protein, a Cas8-like protein. Class 2 systems are generally less commonthan Class 1 systems and are further divided into the following threetypes: Type II systems, which contain the Cas9 protein; Type V systems,which contain Cas12a protein (previously known as Cpf1, and referred toas Cpf1 herein), Cas12b (previously known as C2c1), Cas12c (previouslyknown as C2c3), Cas12d (previously known as CasY), and Cas12e(previously known as CasX); and Type VI systems, which contain Cas13a(previously known as C2c2), Cas13b, and Cas13c. Pyzocha et al., ACSChemical Biology, Vol. 13 (2), pgs. 347-356. In one embodiment, theCRISPR-Cas system for use in the methods provided herein is a Class 2system. In one embodiment, the CRISPR-Cas system for use in the methodsprovided herein is a Type II, Type V or Type VI Class 2 system. In oneembodiment, the CRISPR-Cas system for use in the methods provided hereinis selected from Cas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a,Cas13b, Cas13c or homologs, orthologs or paralogs thereof.

CRISPR systems used in methods disclosed herein comprise a Cas effectormodule comprising one or more nucleic acid guided CRISPR-associated(Cas) nucleases, referred to herein as Cas effector proteins. In someembodiments, the Cas proteins can comprise one or multiple nucleasedomains. A Cas effector protein can target single stranded or doublestranded nucleic acid molecules (e.g. DNA or RNA nucleic acids) and cangenerate double strand or single strand breaks. In some embodiments, theCas effector proteins are wild-type or naturally occurring Cas proteins.In some embodiments, the Cas effector proteins are mutant Cas proteins,wherein one or more mutations, insertions, or deletions are made in a WTor naturally occurring Cas protein (e.g., a parental Cas protein) toproduce a Cas protein with one or more altered characteristics comparedto the parental Cas protein.

In some instances, the Cas protein is a wild-type (WT) nuclease.Non-limiting examples of suitable Cas proteins for use in the presentdisclosure include C2c1, C2c2, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4,Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10,Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm1, Csm2,Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3,Csx17, Csx14, Csx100, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3,Csf4, MAD1-20, SmCsm1, homologes thereof, orthologes thereof, variantsthereof, mutants thereof, or modified versions thereof. Suitable nucleicacid guided nucleases (e.g., Cas 9) can be from an organism from agenus, which includes but is not limited to: Thiomicrospira,Succinivibrio, Candidatus, Porphyromonas, Acidomonococcus, Prevotella,Smithella, Moraxella, Synergistes, Francisella, Leptospira,Catenibacterium, Kandleria, Clostridium, Dorea, Coprococcus,Enterococcus, Fructobacillus, Weissella, Pediococcus, Corynebacter,Sutterella, Legionella, Treponema, Roseburia, Filfactor, Eubacterium,Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola,Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter,Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitralifractor,Mycoplasma, Alicyclobacillus, Brevibacilus, Bacillus, Bacteroidetes,Brevibacilus, Carnobacterium, Clostridiaridium, Clostridium,Desulfonatronum, Desulfovibrio, Helcococcus, Leptotrichia, Listeria,Methanomethyophilus, Methylobacterium, Opitutaceae, Paludibacter,Rhodobacter, Sphaerochaeta, Tuberibacillus, and Campylobacter. Speciesof organism of such a genus can be as otherwise herein discussed.

Suitable nucleic acid guided nucleases (e.g., Cas9) can be from anorganism from a phylum, which includes but is not limited to: Firmicute,Actinobacteria, Bacteroidetes, Proteobacteria, Spirochates, andTenericutes. Suitable nucleic acid guided nucleases can be from anorganism from a class, which includes but is not limited to:Erysipelotrichia, Clostridia, Bacilli, Actinobacteria, Bacteroidetes,Flavobacteria, Alphaproteobacteria, Betaproteobacteria,Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria,Spirochaetes, and Mollicutes. Suitable nucleic acid guided nucleases canbe from an organism from an order, which includes but is not limited to:Clostridiales, Lactobacillales, Actinomycetales, Bacteroidales,Flavobacteriales, Rhizobiales, Rhodospirillales, Burkholderiales,Neisseriales, Legionellales, Nautiliales, Campylobacterales,Spirochaetales, Mycoplasmatales, and Thiotrichales. Suitable nucleicacid guided nucleases can be from an organism from within a family,which includes but is not limited to: Lachnospiraceae, Enterococcaceae,Leuconostocaceae, Lactobacillaceae, Streptococcaceae,Peptostreptococcaceae, Staphylococcaceae, Eubacteriaceae,Corynebacterineae, Bacteroidaceae, Flavobacterium, Cryomoorphaceae,Rhodobiaceae, Rhodospirillaceae, Acetobacteraceae, Sutterellaceae,Neisseriaceae, Legionellaceae, Nautiliaceae, Campylobacteraceae,Spirochaetaceae, Mycoplasmataceae, and Francisellaceae.

Other nucleic acid guided nucleases (e.g., Cas9) suitable for use in themethods, systems, and compositions of the present disclosure includethose derived from an organism such as, but not limited to:Thiomicrospira sp. XS5, Eubacterium rectale, Succinivibriodextrinosolvens, Caandidatus Methanoplasma termitum, CandidatusMethanomethylophilus alvus, Porphyromonas crevioricanis, Flavobacteriumbranchiophilum, Acidomonococcus sp., Lachnospiraceae bacterium COE1,Prevotella brevis ATCC 19188, Smithella sp. SCADC, Moraxella bovoculi,Synergistes jonesii, Bacteroidetes oral taxon 274, Francisellatularensis, Leptospira inadai serovar Lyme str. 10, Acidomonococcus sp.crystal structure (5B43) S. mutans, S. agalactiae, S. equisimilis, S.sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N.tergarcus; S. auricularis, S. carnosus; N. meningitides. N. gonorrhoeae;L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C.sordellii; Francisella tularensis l. Prevotella albensis,Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus,Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacteriumGW2011_GWC2_44_17, Smithella sp. SCADC, Microgenomates, Acidaminococcussp. BV3L6, Lachnospiraceae bacterium MA2020, candidatus Methanoplasmatermitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospirainadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3,Prevotella disiens, Porphvromonas macacae, Catenibacterium sp. CAG:290,Kandleria vitulina, Clostridiales bacterium KA00274, Lachnospiraceaebacterium 3-2, Dorea longicatena, Coprococcus catus GD/7, Enterococcuscolumbae DSM 7374, Fructobacillius sp. EFB-N1, Weissella halotolerans,Pediococcus acidilactici, Lactobacillus curvatus, Streptococcuspyogenes, Lactobacillus versmoldensis, and Filifactor alocis ATCC 35896.See, U.S. Pat. Nos. 8,697,359; 8,771,945; 8,795,965; 8,865,406;8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839;8,993,233; 8,999,641; 9,822,372; 9,840,713; U.S. patent application Ser.No. 13/842,859 (US 2014/0068797 A1); U.S. Pat. Nos. 9,260,723;9,023,649; 9,834,791; 9,637,739; U.S. patent application Ser. No.14/683,443 (US 2015/0240261 A1); U.S. patent application Ser. No.14/743,764 (US 2015/0291961 A1); U.S. Pat. Nos. 9,790,490; 9,688,972;9,580,701; 9,745,562; 9,816,081; 9,677,090; 9,738,687; U.S. applicationSer. No. 15/632,222 (US 2017/0369879 A1); U.S. application Ser. No.15/631,989; U.S. application Ser. No. 15/632,001; and U.S. Pat. No.9,896,696, each of which is herein incorporated by reference.

In some embodiments, a Cas effector protein comprises one or more of thefollowing activities:

a nickase activity, i.e., the ability to cleave a single strand of anucleic acid molecule;

a double stranded nuclease activity, i.e., the ability to cleave bothstrands of a double stranded nucleic acid and create a double strandedbreak;

an endonuclease activity;

an exonuclease activity; and/or

a helicase activity, i.e., the ability to unwind the helical structureof a double stranded nucleic acid.

In aspects of the disclosure the term “guide nucleic acid” refers to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa target sequence (referred to herein as a “targeting segment”) and 2) ascaffold sequence capable of interacting with (either alone or incombination with a tracrRNA molecule) a nucleic acid guided nuclease asdescribed herein (referred to herein as a “scaffold segment”). A guidenucleic acid can be DNA. A guide nucleic acid can be RNA. A guidenucleic acid can comprise both DNA and RNA. A guide nucleic acid cancomprise modified non-naturally occurring nucleotides. In cases wherethe guide nucleic acid comprises RNA, the RNA guide nucleic acid can beencoded by a DNA sequence on a polynucleotide molecule such as aplasmid, linear construct, or editing cassette as disclosed herein.

In some embodiments, the guide nucleic acids described herein are RNAguide nucleic acids (“guide RNAs” or “gRNAs”) and comprise a targetingsegment and a scaffold segment. In some embodiments, the scaffoldsegment of a gRNA is comprised in one RNA molecule and the targetingsegment is comprised in another separate RNA molecule. Such embodimentsare referred to herein as “double-molecule gRNAs” or “two-molecule gRNA”or “dual gRNAs.” In some embodiments, the gRNA is a single RNA moleculeand is referred to herein as a “single-guide RNA” or an “sgRNA.” Theterm “guide RNA” or “gRNA” is inclusive, referring both to two-moleculeguide RNAs and sgRNAs.

The DNA-targeting segment of a gRNA comprises a nucleotide sequence thatis complementary to a sequence in a target nucleic acid sequence. Assuch, the targeting segment of a gRNA interacts with a target nucleicacid in a sequence-specific manner via hybridization (i.e., basepairing), and the nucleotide sequence of the targeting segmentdetermines the location within the target DNA that the gRNA will bind.The degree of complementarity between a guide sequence and itscorresponding target sequence, when optimally aligned using a suitablealignment algorithm, is about or more than about 50%, 60%, 75%, 80%,85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determinedwith the use of any suitable algorithm for aligning sequences. In someembodiments, a guide sequence is about or more than about 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 75, or morenucleotides in length. In some embodiments, a guide sequence is lessthan about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length. Inaspects, the guide sequence is 10-30 nucleotides long. The guidesequence can be 15-20 nucleotides in length. The guide sequence can be15 nucleotides in length. The guide sequence can be 16 nucleotides inlength. The guide sequence can be 17 nucleotides in length. The guidesequence can be 18 nucleotides in length. The guide sequence can be 19nucleotides in length. The guide sequence can be 20 nucleotides inlength.

The scaffold segment of a guide RNA interacts with a one or more Caseffector proteins to form a ribonucleoprotein complex (referred toherein as a CRISPR-RNP or a RNP-complex). The guide RNA directs thebound polypeptide to a specific nucleotide sequence within a targetnucleic acid sequence via the above-described targeting segment. Thescaffold segment of a guide RNA comprises two stretches of nucleotidesthat are complementary to one another and which form a double strandedRNA duplex. Sufficient sequence within the scaffold sequence to promoteformation of a targetable nuclease complex may include a degree ofcomplementarity along the length of two sequence regions within thescaffold sequence, such as one or two sequence regions involved informing a secondary structure. In some cases, the one or two sequenceregions are comprised or encoded on the same polynucleotide. In somecases, the one or two sequence regions are comprised or encoded onseparate polynucleotides. Optimal alignment may be determined by anysuitable alignment algorithm, and may further account for secondarystructures, such as self-complementarity within either the one or twosequence regions. In some embodiments, the degree of complementaritybetween the one or two sequence regions along the length of the shorterof the two when optimally aligned is about or more than about 25%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In someembodiments, at least one of the two sequence regions is about or morethan about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 40, 50, or more nucleotides in length.

A scaffold sequence of a subject gRNA can comprise a secondarystructure. A secondary structure can comprise a pseudoknot region orstem-loop structure. In some examples, the compatibility of a guidenucleic acid and nucleic acid guided nuclease is at least partiallydetermined by sequence within or adjacent to the secondary structureregion of the guide RNA. In some cases, binding kinetics of a guidenucleic acid to a nucleic acid guided nuclease is determined in part bysecondary structures within the scaffold sequence. In some cases,binding kinetics of a guide nucleic acid to a nucleic acid guidednuclease is determined in part by nucleic acid sequence with thescaffold sequence.

A compatible scaffold sequence for a gRNA-Cas effector proteincombination can be found by scanning sequences adjacent to a native Casnuclease loci. In other words, native Cas nucleases can be encoded on agenome within proximity to a corresponding compatible guide nucleic acidor scaffold sequence.

Nucleic acid guided nucleases can be compatible with guide nucleic acidsthat are not found within the nucleases endogenous host. Such orthogonalguide nucleic acids can be determined by empirical testing. Orthogonalguide nucleic acids can come from different bacterial species or besynthetic or otherwise engineered to be non-naturally occurring.Orthogonal guide nucleic acids that are compatible with a common nucleicacid-guided nuclease can comprise one or more common features. Commonfeatures can include sequence outside a pseudoknot region. Commonfeatures can include a pseudoknot region. Common features can include aprimary sequence or secondary structure

A guide nucleic acid can be engineered to target a desired targetsequence by altering the guide sequence such that the guide sequence iscomplementary to the target sequence, thereby allowing hybridizationbetween the guide sequence and the target sequence. A guide nucleic acidwith an engineered guide sequence can be referred to as an engineeredguide nucleic acid. Engineered guide nucleic acids are oftennon-naturally occurring and are not found in nature.

In some embodiments, the present disclosure provides a polynucleotideencoding a gRNA. In some embodiments, a gRNA-encoding nucleic acid iscomprised in an expression vector, e.g., a recombinant expressionvector. In some embodiments, the present disclosure provides apolynucleotide encoding a site-directed modifying polypeptide. In someembodiments, the polynucleotide encoding a site-directed modifyingpolypeptide is comprised in an expression vector, e.g., a recombinantexpression vector.

In some embodiments, the present disclosure provides a gRNA complexedwith a site-directed modifying polypeptide to form an RNP-complex thatis capable of being introduced into a host cell comprising a targetnucleic acid sequence for which the targeting segment of the gRNAcomprising sequence that is complementary thereto. The site-directedmodifying polypeptide can be a nucleic acid guided nuclease. The nucleicacid guided nuclease can be any nucleic acid guided nuclease as known inthe art and/or provided herein (e.g., Cas9). The nucleic acid guidednuclease can be guided by and RNA (e.g., gRNA) and thus be referred toas an RNA guided nuclease or RNA guided endonuclease.

Traditional Methods of Strain Improvement

Traditional approaches to strain improvement can be broadly categorizedinto two types of approaches: directed strain engineering, and randommutagenesis.

Directed engineering methods of strain improvement involve the plannedperturbation of a handful of genetic elements of a specific organism.These approaches are typically focused on modulating specificbiosynthetic or developmental programs, and rely on prior knowledge ofthe genetic and metabolic factors affecting said pathways. In itssimplest embodiments, directed engineering involves the transfer of acharacterized trait (e.g., gene, promoter, or other genetic elementcapable of producing a measurable phenotype) from one organism toanother organism of the same, or different species.

Random approaches to strain engineering involve the random mutagenesisof parent strains, coupled with extensive screening designed to identifyperformance improvements. Approaches to generating these randommutations include exposure to ultraviolet radiation, or mutagenicchemicals such as Ethyl methanesulfonate. Though random and largelyunpredictable, this traditional approach to strain improvement hadseveral advantages compared to more directed genetic manipulations.First, many industrial organisms were (and remain) poorly characterizedin terms of their genetic and metabolic repertoires, renderingalternative directed improvement approaches difficult, if notimpossible.

Second, even in relatively well characterized systems, genotypic changesthat result in industrial performance improvements are difficult topredict, and sometimes only manifest themselves as epistatic phenotypesrequiring cumulative mutations in many genes of known and unknownfunction.

Additionally, for many years, the genetic tools required for makingdirected genomic mutations in a given industrial organism wereunavailable, or very slow and/or difficult to use.

The extended application of the traditional strain improvement programs,however, yield progressively reduced gains in a given strain lineage,and ultimately lead to exhausted possibilities for further strainefficiencies. Beneficial random mutations are relatively rare events,and require large screening pools and high mutation rates. Thisinevitably results in the inadvertent accumulation of many neutraland/or detrimental (or partly detrimental) mutations in “improved”strains, which ultimately create a drag on future efficiency gains.

Another limitation of traditional cumulative improvement approaches isthat little to no information is known about any particular mutation'seffect on any strain metric. This fundamentally limits a researcher'sability to combine and consolidate beneficial mutations, or to removeneutral or detrimental mutagenic “baggage.”

Other approaches and technologies exist to randomly recombine mutationsbetween strains within a mutagenic lineage. For example, some formatsand examples for iterative sequence recombination, sometimes referred toas DNA shuffling, evolution, or molecular breeding, have been describedin U.S. patent application Ser. No. 08/198,431, filed Feb. 17, 1994,Serial No. PCT/US95/02126, filed, Feb. 17, 1995, Ser. No. 08/425,684,filed Apr. 18, 1995, Ser. No. 08/537,874, filed Oct. 30, 1995, Ser. No.08/564,955, filed Nov. 30, 1995, Ser. No. 08/621,859, filed. Mar. 25,1996, Ser. No. 08/621,430, filed Mar. 25, 1996, Serial No.PCT/US96/05480, filed Apr. 18, 1996, Ser. No. 08/650,400, filed May 20,1996, Ser. No. 08/675,502, filed Jul. 3, 1996, Ser. No. 08/721,824,filed Sep. 27, 1996, and Ser. No. 08/722,660 filed Sep. 27, 1996;Stemmer, Science 270:1510 (1995); Stemmer et al., Gene 164:49-53 (1995);Stemmer, Bio/Technology 13:549-553 (1995); Stemmer, Proc. Natl. Acad.Sci. U.S.A. 91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994);Crameri et al., Nature Medicine 2(1):1-3 (1996); Crameri et al., NatureBiotechnology 14:315-319 (1996), each of which is incorporated herein byreference in its entirety for all purposes.

These include techniques such as protoplast fusion and whole genomeshuffling that facilitate genomic recombination across mutated strains.For some industrial microorganisms such as yeast and filamentous fungi,natural mating cycles can also be exploited for pairwise genomicrecombination. In this way, detrimental mutations can be removed by‘back-crossing’ mutants with parental strains and beneficial mutationsconsolidated. Moreover, beneficial mutations from two different strainlineages can potentially be combined, which creates additionalimprovement possibilities over what might be available from mutating asingle strain lineage on its own. However, these approaches are subjectto many limitations that are circumvented using the methods of thepresent disclosure.

For example, traditional recombinant approaches as described above areslow and rely on a relatively small number of random recombinationcrossover events to swap mutations, and are therefore limited in thenumber of combinations that can be attempted in any given cycle, or timeperiod. In addition, although the natural recombination events in theprior art are essentially random, they are also subject to genomepositional bias.

Most importantly, the traditional approaches also provide littleinformation about the influence of individual mutations and due to therandom distribution of recombined mutations many specific combinationscannot be generated and evaluated.

To overcome many of the aforementioned problems associated withtraditional strain improvement programs, the present disclosure setsforth a unique HTP genomic engineering platform that is computationallydriven and integrates molecular biology, automation, data analytics, andmachine learning protocols. This integrative platform utilizes a suiteof HTP molecular tool sets that are used to construct HTP genetic designlibraries. These genetic design libraries will be elaborated upon below.

The presently disclosed HTP platform and its unique microbial geneticdesign libraries fundamentally shift the paradigm of microbial straindevelopment and evolution. For example, traditional mutagenesis-basedmethods of developing an industrial microbial strain will eventuallylead to microbes burdened with a heavy mutagenic load that has beenaccumulated over years of random mutagenesis.

The ability to solve this issue (i.e. remove the genetic baggageaccumulated by these microbes) has eluded microbial researchers fordecades. However, utilizing the HTP platform disclosed herein, theseindustrial strains can be “rehabilitated,” and the genetic mutationsthat are deleterious can be identified and removed. Congruently, thegenetic mutations that are identified as beneficial can be kept, and insome cases improved upon. The resulting microbial strains demonstratesuperior phenotypic traits (e.g., improved production of a compound ofinterest), as compared to their parental strains.

Furthermore, the HTP platform taught herein is able to identify,characterize, and quantify the effect that individual mutations have onmicrobial strain performance. This information, i.e. what effect does agiven genetic change x have on host cell phenotype y (e.g., productionof a compound or product of interest), is able to be generated and thenstored in the microbial HTP genetic design libraries discussed below.That is, sequence information for each genetic permutation, and itseffect on the host cell phenotype are stored in one or more databases,and are available for subsequent analysis (e.g., epistasis mapping, asdiscussed below). The present disclosure also teaches methods ofphysically saving/storing valuable genetic permutations in the form ofgenetic insertion constructs, or in the form of one or more host cellorganisms containing said genetic permutation (e.g., see librariesdiscussed below.)

When one couples these HTP genetic design libraries into an iterativeprocess that is integrated with a sophisticated data analytics andmachine learning process a dramatically different methodology forimproving host cells emerges. The taught platform is thereforefundamentally different from the previously discussed traditionalmethods of developing host cell strains. The taught HTP platform doesnot suffer from many of the drawbacks associated with the previousmethods. These and other advantages will become apparent with referenceto the HTP molecular tool sets and the derived genetic design librariesdiscussed below.

Overview

It is an object of the present disclosure to circumvent all thelimitations described above by providing a high-throughput method fortransforming filamentous fungal cells or protoplasts derived therefrom,purifying homokaryotic transformants and screening purifiedtransformants. In general, the methods and systems described hereinentail preparation of protoplasts from filamentous fungal cells,transformation of the prepared protoplasts, purification of protoplastscontaining a single nucleus by altering the growth conditions used toprepare mycelia for protoplast preparation. Strain purification isachieved through selection and counter-selection, and, optionally,screening purified transformants possessing the correct phenotype and/orproducing products of interest. The products of interest can be producedat a desired yield, productivity or titer. Preferably, protoplasts areused, but the method is applicable to other fungal cell types. In somecases, the methods and systems provided herein are high-throughput. Insome cases, the methods and systems provided herein comprise steps thatare semi-automated (e.g., transformation or selection,counterselection). In some cases, the methods and systems providedherein comprise steps that are fully automated. In some cases, themethods and systems provided herein are high-throughput and the stepstherein are semi-automated (e.g., transformation or selection,counterselection) or fully automated. As used herein, high-throughputcan refer to any partially—or fully-automated method provided hereinthat is capable of evaluating about 1,000 or more transformants per day,and particularly to those methods capable of evaluating 5,000 or moretransformants per day, and most particularly to methods capable ofevaluating 10,000 or more transformants per day. Moreover, suitablevolumes in which the method is performed are those of commerciallyavailable (deep well) microtiter plates, i.e. smaller than 1 ml,preferably smaller than 500 ul, more preferably smaller than 250 ul,most preferably from 1.5 ul to 250 ul, still most preferably from 10 ulto 100 ul.

The filamentous fungal cells used to prepare the protoplasts can be anyfilamentous fungus strains known in the art or described hereinincluding holomorphs, teleomorphs or anamorphs thereof. The preparationof the protoplasts can be performed using those described herein or anyknown method in the art for preparing protoplasts.

Transformation of the protoplasts can be with at least onepolynucleotide designed to integrate into a pre-determined locus in thefilamentous fungal genome as provided herein. In a preferred embodiment,the protoplasts are co-transformed with at least two polynucleotides asprovided herein such that each polynucleotide construct is designed tointegrate into a different pre-determined locus in the filamentousfungal genome. A pre-determined locus can be for a target filamentousfungal gene (e.g., a gene whose protein product is involved in citricacid production) or a selectable marker gene present in the filamentousfungal genome. A polynucleotide for use in transforming orco-transforming protoplasts using the methods or systems provided hereincan comprise sequence of a target filamentous fungal gene (e.g., a genewhose protein product is involved in citric acid production) comprisingor containing a mutation and/or a genetic control element(s). Themutation can be a small nuclear polymorphism(s) such as a singlenucleotide polymorphism, sequence insertions, deletions, inversions, andother sequence replacements. The genetic control element can be apromoter sequence (endogenous or heterologous) and/or a terminatorsequence (endogenous or heterologous). The promoter can be inducible. Apolynucleotide for use in transforming or co-transforming protoplastsusing the methods or systems provided herein can comprise sequence of aselectable marker gene. A polynucleotide for use in transforming orco-transforming protoplasts using the methods or systems provided hereincan be separated into two or more portions such that integration of thewhole polynucleotide in a transformed protoplast occurs only if eachseparate portion of the polynucleotide integrates at the same targetsite in the transformed protoplast's genome. Each portion of thepolynucleotide can comprise a mutation and/or genetic control element asprovided herein. In one embodiment, the methods and systems providedherein entail co-transformation of protoplasts provided herein with twopolynucleotides such that a first polynucleotide comprise sequence of atarget filamentous fungal gene (e.g., a gene whose protein product isinvolved in citric acid production) comprising or containing a mutationand/or a genetic control element(s), while a second polynucleotidecomprises sequence of a selectable marker gene. Further to thisembodiment, the second polynucleotide can be designed to integrate intoan additional selectable marker gene in the protoplast genome, while thefirst polynucleotide can be designed to integrate into the locus for thetarget filamentous fungal gene or, alternatively, into the locus of yeta further selectable marker gene. A selectable marker gene in any of theembodiments provided herein can be any of the selectable marker genesdescribed herein.

It is also the object of this disclosure to provide a method forpreparing and storing a plurality of protoplasts from filamentous fungalcells. The method can entail removing cell walls from the filamentousfungal cells in the fungal culture, isolating the protoplasts, andresuspending the isolated protoplasts in a mixture comprising at leastdimethyl sulfoxide (DMSO) and storing the isolated protoplasts. Storagecan be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 24 hours.Storage can be for at least 1, 7, 14, 30 or more days. Storage can befor at least 3, 6, 12, or more months. Storage can be at 4, −20 or −80°C. The fungal culture can be a culture with a volume of at least 500 ml,1 liter, 2 liters, 3 liters, 4 liters or 5 liters. The filamentousfungal cells can be any filamentous fungus provided herein or known inthe art. Prior to preparation of the protoplasts the fungal culture canbe grown for at least 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours. Inone embodiment, the fungal culture is grown under conditions whereby atleast 70% of the protoplasts are homokaryotic following preparation ofthe protoplasts. In another embodiment, removing the cell walls isperformed by enzymatic digestion. The enzymatic digestion can beperformed with mixture of enzymes comprising a beta-glucanase and apolygalacturonase. The enzymatic digestion can be performed withVinoTaste concentrate. In yet another embodiment, the method furthercomprises adding polyethylene glycol (PEG) to the mixture comprisingDMSO prior to storing the protoplasts. The PEG can be added to a finalconcentration of 50%, 40%, 30%, 20%, 15%, 10%, 5% or less. In stillanother embodiment, the method further comprises distributing theprotoplasts into microtiter plates prior to storing the protoplasts. Themicrotiter plate can be a 6 well, 12 well, 24 well, 96 well, 384 well or1536 well plate.

Genetic Design & Microbial Engineering: A Systematic CombinatorialApproach to Strain Improvement Utilizing a Suite of HTP Molecular Toolsand HTP Genetic Design Libraries

As aforementioned, the present disclosure provides a novel HTP platformand genetic design strategy for engineering microbial organisms throughiterative systematic introduction and removal of genetic changes acrossstrains. The platform is supported by a suite of molecular tools, whichenable the creation of HTP genetic design libraries and allow for theefficient implementation of genetic alterations into a given hoststrain.

The HTP genetic design libraries of the disclosure serve as sources ofpossible genetic alterations that may be introduced into a particularmicrobial (e.g., filamentous fungal) strain background. In this way, theHTP genetic design libraries are repositories of genetic diversity, orcollections of genetic perturbations, which can be applied to theinitial or further engineering of a given microbial strain. Techniquesfor programming genetic designs for implementation to host strains aredescribed in pending U.S. patent application Ser. No. 15/140,296 andpending International Application Serial No. PCT/US17/29725, entitled“Microbial Strain Design System and Methods for Improved Large ScaleProduction of Engineered Nucleotide Sequences,” each of which isincorporated by reference in its entirety herein.

The HTP molecular tool sets utilized in this platform may include, interalia: (1) Promoter swaps (PRO Swap), (2) SNP swaps, (3) Start/Stop codonexchanges, (4) STOP swaps, and (5) Sequence optimization. The HTPmethods of the present disclosure also teach methods for directing theconsolidation/combinatorial use of HTP tool sets, including (6)Epistasis mapping protocols. As aforementioned, this suite of moleculartools, either in isolation or combination, enables the creation of HTPgenetic design host cell libraries.

As will be demonstrated, utilization of the aforementioned HTP geneticdesign libraries in the context of the taught HTP microbial engineeringplatform enables the identification and consolidation of beneficial“causative” mutations or gene sections and also the identification andremoval of passive or detrimental mutations or gene sections. This newapproach allows rapid improvements in strain performance that could notbe achieved by traditional random mutagenesis or directed geneticengineering. The removal of genetic burden or consolidation ofbeneficial changes into a strain with no genetic burden also provides anew, robust starting point for additional random mutagenesis that mayenable further improvements.

In some embodiments, the present disclosure teaches that as orthogonalbeneficial changes are identified across various discrete branches of amutagenic strain lineage, they can also be rapidly consolidated intobetter performing strains. These mutations can also be consolidated intostrains that are not part of mutagenic lineages, such as strains withimprovements gained by directed genetic engineering.

In some embodiments, the present disclosure differs from known strainimprovement approaches in that it analyzes the genome-wide combinatorialeffect of mutations across multiple disparate genomic regions, includingexpressed and non-expressed genetic elements, and uses gatheredinformation (e.g., experimental results) to predict mutationcombinations expected to produce strain enhancements.

In some embodiments, the present disclosure teaches: i) industrialmicroorganisms, and other host cells amenable to improvement via thedisclosed disclosures, ii) generating diversity pools for downstreamanalysis, iii) methods and hardware for high-throughput screening andsequencing of large variant pools, iv) methods and hardware for machinelearning computational analysis and prediction of synergistic effects ofgenome-wide mutations, and v) methods for high-throughput strainengineering.

The following molecular tools and libraries are discussed in terms ofillustrative microbial examples. Persons having skill in the art willrecognize that the HTP molecular tools of the present disclosure arecompatible with any host cell, including eukaryotic cellular, and higherlife forms such as, for example, the same principles and process can bedeployed in filamentous fungal cells (e.g., Aspergillus niger).

Each of the identified HTP molecular tool sets-which enable the creationof the various HTP genetic design libraries utilized in the microbialengineering platform-will now be discussed.

1. Promoter Swaps: A Molecular Tool for the Derivation of Promoter SwapMicrobial Strain Libraries

In some embodiments, the present disclosure teaches methods of selectingpromoters with optimal expression properties to produce beneficialeffects on overall-host strain phenotype (e.g., yield or productivity).

For example, in some embodiments, the present disclosure teaches methodsof identifying one or more promoters and/or generating variants of oneor more promoters within a host cell, which exhibit a range ofexpression strengths (e.g. promoter ladders discussed infra), orsuperior regulatory properties (e.g., tighter regulatory control forselected genes). A particular combination of these identified and/orgenerated promoters can be grouped together as a promoter ladder, whichis explained in more detail below.

The promoter ladder in question is then associated with a given gene ofinterest. Thus, if one has promoters P₁-P₈ (representing eight promotersthat have been identified and/or generated to exhibit a range ofexpression strengths) and associates the promoter ladder with a singlegene of interest in a microbe (i.e. genetically engineer a microbe witha given promoter operably linked to a given target gene), then theeffect of each combination of the eight promoters can be ascertained bycharacterizing each of the engineered strains resulting from eachcombinatorial effort, given that the engineered microbes have anotherwise identical genetic background except the particular promoter(s)associated with the target gene.

The resultant microbes that are engineered via this process form HTPgenetic design libraries.

In a specific embodiment, the promoter swapping (PRO Swap) methodsprovided herein entail systematically associating each promoter from thepromoter ladder depicted in Table 1 with a gene shown to or suspected toplay a role or affect morphology of filamentous fungal cells when grownunder specific conditions (referred to as target morphological genes).The perturbation of the gene can cause a desired morphologicalphenotype. The desired phenotype can be a non-mycelium, pelletmorphology when grown in submerged cultures of a production media (e.g.,CAP media). Thus, if one has promoters P₁-P₄ (representing the fourpromoters from Table 1 that have been identified and/or generated toexhibit a range of expression strengths) and associates the promoterladder with a single target morphological gene of interest in a microbe(i.e. genetically engineer a microbe with a given promoter operablylinked to a given target morphological gene), then the effect of eachcombination of the four promoters can be ascertained by characterizingeach of the engineered strains resulting from each combinatorial effort,given that the engineered microbes have an otherwise identical geneticbackground except the particular promoter(s) associated with thespecific target morphological gene. The resultant microbes that areengineered via this process can form HTP morphological genetic designlibraries. The gene shown to or suspected to play a role or affectmorphology of filamentous fungal cells can be any such gene known in theart and/or provided herein.

The HTP genetic design library can refer to the actual physicalmicrobial strain collection that is formed via this process, with eachmember strain being representative of a given promoter operably linkedto a particular target gene, in an otherwise identical geneticbackground, said library being termed a “promoter swap microbial strainlibrary.” In the specific context of filamentous fungi (e.g., A. niger),the library can be termed a “promoter swap filamentous fungal strainlibrary,” or “promoter swap A. niger strain library,” but the terms canbe used synonymously, as filamentous fungus or A. niger are specificexamples of a microbe or coenocytic organism.

Furthermore, the HTP genetic design library can refer to the collectionof genetic perturbations—in this case a given promoter x operably linkedto a given gene y—said collection being termed a “promoter swaplibrary.”

Further, one can utilize the same promoter ladder comprising promotersP₁-P₈ to engineer microbes, wherein each of the 8 promoters is operablylinked to 10 different gene targets. The result of this procedure wouldbe 80 microbes that are otherwise assumed genetically identical, exceptfor the particular promoters operably linked to a target gene ofinterest. These 80 microbes could be appropriately screened andcharacterized and give rise to another HTP genetic design library. Thecharacterization of the microbial strains in the HTP genetic designlibrary produces information and data that can be stored in any datastorage construct, including a relational database, an object-orienteddatabase or a highly distributed NoSQL database. This data/informationcould be, for example, a given promoter's (e.g. P₁-P₈) effect whenoperably linked to a given gene target. This data/information can alsobe the broader set of combinatorial effects that result from operablylinking two or more of promoters P₁-P₈ to a given gene target.

The aforementioned examples of eight promoters and 10 target genes ismerely illustrative, as the concept can be applied with any given numberof promoters that have been grouped together based upon exhibition of arange of expression strengths and any given number of target genes.Persons having skill in the art will also recognize the ability tooperably link two or more promoters in front of any gene target. Thus,in some embodiments, the present disclosure teaches promoter swaplibraries in which 1, 2, 3 or more promoters from a promoter ladder areoperably linked to one or more genes.

In summary, utilizing various promoters to drive expression of variousgenes in an organism is a powerful tool to optimize a trait of interest.The molecular tool of promoter swapping, developed by the inventors,uses a ladder of promoter sequences (e.g., Table 1) that have beendemonstrated to vary expression of at least one locus under at least onecondition. This ladder is then systematically applied to a group ofgenes (e.g., within the same pathway as FungiSNP_18 as provided herein)in the organism using high-throughput genome engineering. This group ofgenes is determined to have a high likelihood of impacting the trait ofinterest based on any one of a number of methods. These could includeselection based on known function, or impact on the trait of interest,or algorithmic selection based on previously determined beneficialgenetic diversity. In some embodiments, the selection of genes caninclude all the genes in a given host. In other embodiments, theselection of genes can be a subset of all genes in a given host, chosenrandomly.

The resultant HTP genetic design microbial strain library of organismscontaining a promoter sequence linked to a gene is then assessed forperformance in a high-throughput screening model, and promoter-genelinkages which lead to increased performance are determined and theinformation stored in a database. The collection of geneticperturbations (i.e. given promoter x operably linked to a given gene y)form a “promoter swap library,” which can be utilized as a source ofpotential genetic alterations to be utilized in microbial engineeringprocessing. Over time, as a greater set of genetic perturbations isimplemented against a greater diversity of host cell backgrounds, eachlibrary becomes more powerful as a corpus of experimentally confirmeddata that can be used to more precisely and predictably design targetedchanges against any background of interest.

Transcription levels of genes in an organism are a key point of controlfor affecting organism behavior. Transcription is tightly coupled totranslation (protein expression), and which proteins are expressed inwhat quantities determines organism behavior. Cells express thousands ofdifferent types of proteins, and these proteins interact in numerouscomplex ways to create function. By varying the expression levels of aset of proteins systematically, function can be altered in ways that,because of complexity, are difficult to predict. Some alterations mayincrease performance, and so, coupled to a mechanism for assessingperformance, this technique allows for the generation of organisms withimproved function.

In the context of a small molecule synthesis pathway, enzymes interactthrough their small molecule substrates and products in a linear orbranched chain, starting with a substrate and ending with a smallmolecule of interest. Because these interactions are sequentiallylinked, this system exhibits distributed control, and increasing theexpression of one enzyme can only increase pathway flux until anotherenzyme becomes rate limiting.

Metabolic Control Analysis (MCA) is a method for determining, fromexperimental data and first principles, which enzyme or enzymes are ratelimiting. MCA is limited however, because it requires extensiveexperimentation after each expression level change to determine the newrate limiting enzyme. Promoter swapping is advantageous in this context,because through the application of a promoter ladder to each enzyme in apathway, the limiting enzyme is found, and the same thing can be done insubsequent rounds to find new enzymes that become rate limiting.Further, because the read-out on function is better production of thesmall molecule of interest, the experiment to determine which enzyme islimiting is the same as the engineering to increase production, thusshortening development time. In some embodiments the present disclosureteaches the application of PRO swap to genes encoding individualsubunits of multi-unit enzymes. In yet other embodiments, the presentdisclosure teaches methods of applying PRO swap techniques to genesresponsible for regulating individual enzymes, or whole biosyntheticpathways.

In some embodiments, the promoter swap tool of the present disclosure isused to identify optimum expression of a selected gene target. In someembodiments, the goal of the promoter swap may be to increase expressionof a target gene to reduce bottlenecks in a metabolic or geneticpathway. In other embodiments, the goal of the promoter swap may be toreduce the expression of the target gene to avoid unnecessary energyexpenditures in the host cell, when expression of said target gene isnot required.

In the context of other cellular systems like transcription, transport,or signaling, various rational methods can be used to try and find out,apriori, which proteins are targets for expression change and what thatchange should be. These rational methods reduce the number ofperturbations that must be tested to find one that improves performance,but they do so at significant cost. Gene deletion studies identifyproteins whose presence is critical for a particular function, andimportant genes can then be over-expressed. Due to the complexity ofprotein interactions, this is often ineffective at increasingperformance. Different types of models have been developed that attemptto describe, from first principles, transcription or signaling behavioras a function of protein levels in the cell. These models often suggesttargets where expression changes might lead to different or improvedfunction. The assumptions that underlie these models are simplistic andthe parameters difficult to measure, so the predictions they make areoften incorrect, especially for non-model organisms. With both genedeletion and modeling, the experiments required to determine how toaffect a certain gene are different than the subsequent work to make thechange that improves performance. Promoter swapping sidesteps thesechallenges, because the constructed strain that highlights theimportance of a particular perturbation is also, already, the improvedstrain.

Thus, in particular embodiments, promoter swapping is a multi-stepprocess comprising:

1. Selecting a set of “x” promoters to act as a “ladder.” Ideally thesepromoters have been shown to lead to highly variable expression acrossmultiple genomic loci, but the only requirement is that they perturbgene expression in some way.

2. Selecting a set of “n” genes to target. This set can be every openreading frame (ORF) in a genome, or a subset of ORFs. The subset can bechosen using annotations on ORFs related to function, by relation topreviously demonstrated beneficial perturbations (previous promoterswaps or previous SNP swaps), by algorithmic selection based onepistatic interactions between previously generated perturbations, otherselection criteria based on hypotheses regarding beneficial ORF totarget, or through random selection. In other embodiments, the “n”targeted genes can comprise non-protein coding genes, includingnon-coding RNAs. In one embodiment, the set of “n” genes can beorthologues of the S. cerevisiae SLN1 gene and orthologues of one ormore genes that are part of the same pathway. The orthologues of the S.cerevisiae SLN1 gene and one or more genes that are part of the samepathway can be wild-type are mutant forms of said genes. In oneembodiment, the filamentous fungal strain or host cell is A. niger, andthe set of “n” genes selected is the SNPs in Table 4. In anotherembodiment wherein A. niger is the host cell, the set of “n” genesselected is the non-SNPs or wildtype versions of the SNP containinggenes in Table 4. When A. niger is the host cell, the set of “n” genescan be the gene for FungiSNP_9 found in Table 4 in addition to one ormore genes that are part of the same pathway. When A. niger is the hostcell, the set of “n” genes can be the gene for FungiSNP_12 found inTable 4 in addition to one or more genes that are part of the samepathway. When A. niger is the host cell, the set of “n” genes can be thegene for FungiSNP_40 found in Table 4 in addition to one or more genesthat are part of the same pathway. In a preferred embodiment, when A.niger is the host cell, the set of “n” genes can be the gene forFungiSNP_18 (i.e., a mutant form of the A. niger orthologue of the S.cerevisiae SLN1 gene) from Table 4 in addition to one or more genes thatare part of the same pathway. The A. niger orthologue of the S.cerevisiae SLN1 gene and/or the one or more genes in the same pathwaycan be wild-type or mutant forms of the gene. A mutant form of the A.niger orthologue of the S. cerevisiae SLN1 gene can be the form with SEQID NO: 13. The one or more genes in the pathway can be an A. nigerorthologue of the S. cerevisiae Ypd1, Skn7, Ssk1 and Ssk2 genes or anycombination thereof. The one or more genes that are part of the samepathway can be selected from the nucleic acid sequences represented bySEQ ID NOs: 15, 16, 17, 18, 19 or any combination thereof.

3. High-throughput strain engineering to rapidly—and in someembodiments, in parallel-carry out the following genetic modifications:When a native promoter exists in front of target gene n and its sequenceis known, replace the native promoter with each of the x promoters inthe ladder. When the native promoter does not exist, or its sequence isunknown, insert each of the x promoters in the ladder in front of gene n(see e.g., FIG. 10). In this way a “library” (also referred to as a HTPgenetic design library) of strains is constructed, wherein each memberof the library is an instance of x promoter operably linked to n target,in an otherwise identical genetic context. As previously describedcombinations of promoters can be inserted, extending the range ofcombinatorial possibilities upon which the library is constructed.

4. High-throughput screening of the library of strains in a contextwhere their performance against one or more metrics is indicative of theperformance that is being optimized.

This foundational process can be extended to provide furtherimprovements in strain performance by, inter alia: (1) Consolidatingmultiple beneficial perturbations into a single strain background,either one at a time in an interactive process, or as multiple changesin a single step. Multiple perturbations can be either a specific set ofdefined changes or a partly randomized, combinatorial library ofchanges. For example, if the set of targets is every gene in a pathway,then sequential regeneration of the library of perturbations into animproved member or members of the previous library of strains canoptimize the expression level of each gene in a pathway regardless ofwhich genes are rate limiting at any given iteration; (2) Feeding theperformance data resulting from the individual and combinatorialgeneration of the library into an algorithm that uses that data topredict an optimum set of perturbations based on the interaction of eachperturbation; and (3) Implementing a combination of the above twoapproaches.

The molecular tool, or technique, discussed above is characterized aspromoter swapping, but is not limited to promoters and can include othersequence changes that systematically vary the expression level of a setof targets. Other methods for varying the expression level of a set ofgenes could include: a) a ladder of ribosome binding sites (or Kozaksequences in eukaryotes); b) replacing the start codon of each targetwith each of the other start codons (i.e start/stop codon exchangesdiscussed infra); c) attachment of various mRNA stabilizing ordestabilizing sequences to the 5′ or 3′ end, or at any other location,of a transcript, d) attachment of various protein stabilizing ordestabilizing sequences at any location in the protein.

The approach is exemplified in the present disclosure with industrialmicroorganisms, but is applicable to any organism where desired traitscan be identified in a population of genetic mutants. For example, thiscould be used for improving the performance of CHO cells, yeast, insectcells, algae, as well as multi-cellular organisms, such as plants.

2. SNP Swaps: A Molecular Tool for the Derivation of SNP Swap MicrobialStrain Libraries

In certain embodiments, SNP swapping is not a random mutagenic approachto improving a microbial strain, but rather involves the systematicintroduction or removal of individual Small Nuclear Polymorphismnucleotide mutations (i.e. SNPs) (hence the name “SNP swapping”) acrossstrains. FIG. 9 conceptually depicts a round of SNP Swapping in thefilamentous fungal cells of the present invention. The demonstration ofthe utility of SNP swapping in filamentous fungal cells is shown in FIG.42.

In one embodiment, the methods and systems provided herein are utilizedfor SNP swapping in order to generate filamentous fungal librariescomprising filamentous fungi with individual SNPs or combinations ofSNPs. Combinatorial SNP swapping can be achieved using bipartitetransformation as illustrated in FIG. 37.

The resultant microbes that are engineered via this process form HTPgenetic design libraries.

The HTP genetic design library can refer to the actual physicalmicrobial strain collection that is formed via this process, with eachmember strain being representative of the presence or absence of a givenSNP, in an otherwise identical genetic background, said library beingtermed a “SNP swap microbial strain library.” In the specific context offilamentous fungus (e.g., A. niger), the library can be termed a “SNPswap filamentous fungal strain library,” or “SNP swap A. niger strainlibrary,” but the terms can be used synonymously, as filamentous fungusis a specific example of a microbe or coenocytic organism.

Furthermore, the HTP genetic design library can refer to the collectionof genetic perturbations—in this case a given SNP being present or agiven SNP being absent-said collection being termed a “SNP swaplibrary.”

In some embodiments, SNP swapping involves the reconstruction of hostorganisms with optimal combinations of target SNP “building blocks” withidentified beneficial performance effects. Thus, in some embodiments,SNP swapping involves consolidating multiple beneficial mutations into asingle strain background, either one at a time in an iterative process,or as multiple changes in a single step. Multiple changes can be eithera specific set of defined changes or a partly randomized, combinatoriallibrary of mutations.

In other embodiments, SNP swapping also involves removing multiplemutations identified as detrimental from a strain, either one at a timein an iterative process, or as multiple changes in a single step.Multiple changes can be either a specific set of defined changes or apartly randomized, combinatorial library of mutations. In someembodiments, the SNP swapping methods of the present disclosure includeboth the addition of beneficial SNPs, and removing detrimental and/orneutral mutations.

SNP swapping is a powerful tool to identify and exploit both beneficialand detrimental mutations in a lineage of strains subjected tomutagenesis and selection for an improved trait of interest. SNPswapping utilizes high-throughput genome engineering techniques tosystematically determine the influence of individual mutations in amutagenic lineage. Genome sequences are determined for strains acrossone or more generations of a mutagenic lineage with known performanceimprovements. High-throughput genome engineering is then usedsystematically to recapitulate mutations from improved strains inearlier lineage strains, and/or revert mutations in later strains toearlier strain sequences. The performance of these strains is thenevaluated and the contribution of each individual mutation on theimproved phenotype of interest can be determined. As aforementioned, themicrobial strains that result from this process areanalyzed/characterized and form the basis for the SNP swap geneticdesign libraries that can inform microbial strain improvement acrosshost strains.

Removal of detrimental mutations can provide immediate performanceimprovements, and consolidation of beneficial mutations in a strainbackground not subject to mutagenic burden can rapidly and greatlyimprove strain performance. The various microbial strains produced viathe SNP swapping process form the HTP genetic design SNP swappinglibraries, which are microbial strains comprising the variousadded/deleted/or consolidated SNPs, but with otherwise identical geneticbackgrounds.

As discussed previously, random mutagenesis and subsequent screening forperformance improvements is a commonly used technique for industrialstrain improvement, and many strains currently used for large scalemanufacturing have been developed using this process iteratively over aperiod of many years, sometimes decades. Random approaches to generatinggenomic mutations such as exposure to UV radiation or chemical mutagenssuch as ethyl methanesulfonate were a preferred method for industrialstrain improvements because: 1) industrial organisms may be poorlycharacterized genetically or metabolically, rendering target selectionfor directed improvement approaches difficult or impossible; 2) even inrelatively well characterized systems, changes that result in industrialperformance improvements are difficult to predict and may requireperturbation of genes that have no known function, and 3) genetic toolsfor making directed genomic mutations in a given industrial organism maynot be available or very slow and/or difficult to use.

However, despite the aforementioned benefits of this process, there arealso a number of known disadvantages. Beneficial mutations arerelatively rare events, and in order to find these mutations with afixed screening capacity, mutations rates must be sufficiently high.This often results in unwanted neutral and partly detrimental mutationsbeing incorporated into strains along with beneficial changes. Over timethis ‘mutagenic burden’ builds up, resulting in strains withdeficiencies in overall robustness and key traits such as growth rates.Eventually ‘mutagenic burden’ renders further improvements inperformance through random mutagenesis increasingly difficult orimpossible to obtain. Without suitable tools, it is impossible toconsolidate beneficial mutations found in discrete and parallel branchesof strain lineages.

SNP swapping is an approach to overcome these limitations bysystematically recapitulating or reverting some or all mutationsobserved when comparing strains within a mutagenic lineage. In this way,both beneficial (‘causative’) mutations can be identified andconsolidated, and/or detrimental mutations can be identified andremoved. This allows rapid improvements in strain performance that couldnot be achieved by further random mutagenesis or targeted geneticengineering.

Removal of genetic burden or consolidation of beneficial changes into astrain with no genetic burden also provides a new, robust starting pointfor additional random mutagenesis that may enable further improvements.

In addition, as orthogonal beneficial changes are identified acrossvarious, discrete branches of a mutagenic strain lineage, they can berapidly consolidated into better performing strains. These mutations canalso be consolidated into strains that are not part of mutageniclineages, such as strains with improvements gained by directed geneticengineering.

Other approaches and technologies exist to randomly recombine mutationsbetween strains within a mutagenic lineage. These include techniquessuch as protoplast fusion and whole genome shuffling that facilitategenomic recombination across mutated strains. For some industrialmicroorganisms such as yeast and filamentous fungi, natural matingcycles can also be exploited for pairwise genomic recombination. In thisway, detrimental mutations can be removed by ‘back-crossing’ mutantswith parental strains and beneficial mutations consolidated. However,these approaches are subject to many limitations that are circumventedusing the SNP swapping methods of the present disclosure.

For example, as these approaches rely on a relatively small number ofrandom recombination crossover events to swap mutations, it may takemany cycles of recombination and screening to optimize strainperformance. In addition, although natural recombination events areessentially random, they are also subject to genome positional bias andsome mutations may be difficult to address. These approaches alsoprovide little information about the influence of individual mutationswithout additional genome sequencing and analysis. SNP swappingovercomes these fundamental limitations as it is not a random approach,but rather the systematic introduction or removal of individualmutations across strains.

In some embodiments, the present disclosure teaches methods foridentifying the SNP sequence diversity present among the organisms of adiversity pool. A diversity pool can be a given number in of microbesutilized for analysis, with said microbes' genomes representing the“diversity pool.”

In particular aspects, a diversity pool may be an original parent strain(S₁) with a “baseline” or “reference” genetic sequence at a particulartime point (S₁Gen₁) and then any number of subsequent offspring strains(S_(2-n)) that were derived/developed from said S₁ strain and that havea different genome (S_(2-n)Gen_(2-n)), in relation to the baselinegenome of S₁.

For example, in some embodiments, the present disclosure teachessequencing the microbial genomes in a diversity pool to identify theSNPs present in each strain. In one embodiment, the strains of thediversity pool are historical microbial production strains. Thus, adiversity pool of the present disclosure can include for example, anindustrial reference strain, and one or more mutated industrial strainsproduced via traditional strain improvement programs.

In some embodiments, the SNPs within a diversity pool are determinedwith reference to a “reference strain.” In some embodiments, thereference strain is a wild-type strain. In other embodiments, thereference strain is an original industrial strain prior to beingsubjected to any mutagenesis. The reference strain can be defined by thepractitioner and does not have to be an original wild-type strain ororiginal industrial strain. The base strain is merely representative ofwhat will be considered the “base,” “reference” or original geneticbackground, by which subsequent strains that were derived, or weredeveloped from said reference strain, are to be compared.

Once all SNPS in the diversity pool are identified, the presentdisclosure teaches methods of SNP swapping and screening methods todelineate (i.e. quantify and characterize) the effects (e.g. creation ofa phenotype of interest) of SNPs individually and/or in groups.

In some embodiments, the SNP swapping methods of the present disclosurecomprise the step of introducing one or more SNPs identified in amutated strain (e.g., a strain from amongst S_(2-n)Gen_(2-n)) to areference strain (S₁Gen₁) or wild-type strain (“wave up”).

In other embodiments, the SNP swapping methods of the present disclosurecomprise the step of removing one or more SNPs identified in a mutatedstrain (e.g., a strain from amongst S_(2-n)Gen_(2-n)) (“wave down”).

In some embodiments, each generated strain comprising one or more SNPchanges (either introducing or removing) is cultured and analyzed underone or more criteria of the present disclosure (e.g., production of achemical or product of interest). Data from each of the analyzed hoststrains is associated, or correlated, with the particular SNP, or groupof SNPs present in the host strain, and is recorded for future use.Thus, the present disclosure enables the creation of large and highlyannotated HTP genetic design microbial strain libraries that are able toidentify the effect of a given SNP on any number of microbial genetic orphenotypic traits of interest. The information stored in these HTPgenetic design libraries informs the machine learning algorithms of theHTP genomic engineering platform and directs future iterations of theprocess, which ultimately leads to evolved microbial organisms thatpossess highly desirable properties/traits.

3. Start/Stop Codon Exchanges: A Molecular Tool for the Derivation ofStart/Stop Codon Microbial Strain Libraries

In some embodiments, the present disclosure teaches methods of swappingstart and stop codon variants. For example, typical stop codons for S.cerevisiae and mammals are TAA (UAA) and TGA (UGA), respectively. Thetypical stop codon for monocotyledonous plants is TGA (UGA), whereasinsects and E. coli commonly use TAA (UAA) as the stop codon (Dalphin etal. (1996) Nucl. Acids Res. 24: 216-218). In other embodiments, thepresent disclosure teaches use of the TAG (UAG) stop codons.

The present disclosure similarly teaches swapping start codons. In someembodiments, the present disclosure teaches use of the ATG (AUG) startcodon utilized by most organisms (especially eukaryotes). In someembodiments, the present disclosure teaches that prokaryotes use ATG(AUG) the most, followed by GTG (GUG) and TTG (UUG).

In other embodiments, the present disclosure teaches replacing ATG startcodons with TTG. In some embodiments, the present disclosure teachesreplacing ATG start codons with GTG. In some embodiments, the presentdisclosure teaches replacing GTG start codons with ATG. In someembodiments, the present disclosure teaches replacing GTG start codonswith TTG. In some embodiments, the present disclosure teaches replacingTTG start codons with ATG. In some embodiments, the present disclosureteaches replacing TTG start codons with GTG.

In other embodiments, the present disclosure teaches replacing TAA stopcodons with TAG. In some embodiments, the present disclosure teachesreplacing TAA stop codons with TGA. In some embodiments, the presentdisclosure teaches replacing TGA stop codons with TAA. In someembodiments, the present disclosure teaches replacing TGA stop codonswith TAG. In some embodiments, the present disclosure teaches replacingTAG stop codons with TAA. In some embodiments, the present disclosureteaches replacing TAG stop codons with TGA.

4. Stop Swap: A Molecular Tool for the Derivation of STOP Swap MicrobialStrain Libraries

In some embodiments, the present disclosure teaches methods of improvinghost cell productivity through the optimization of cellular genetranscription. Gene transcription is the result of several distinctbiological phenomena, including transcriptional initiation (RNAprecruitment and transcriptional complex formation), elongation (strandsynthesis/extension), and transcriptional termination (RNAp detachmentand termination). Although much attention has been devoted to thecontrol of gene expression through the transcriptional modulation ofgenes (e.g., by changing promoters, or inducing regulatory transcriptionfactors), comparatively few efforts have been made towards themodulation of transcription via the modulation of gene terminatorsequences.

The most obvious way that transcription impacts on gene expressionlevels is through the rate of Pol II initiation, which can be modulatedby combinations of promoter or enhancer strength and trans-activatingfactors (Kadonaga, J T. 2004 “Regulation of RNA polymerase IItranscription by sequence-specific DNA binding factors” Cell. 2004 Jan.23; 116(2):247-57). In eukaryotes, elongation rate may also determinegene expression patterns by influencing alternative splicing (Cramer P.et al., 1997 “Functional association between promoter structure andtranscript alternative splicing.” Proc Natl Acad Sci USA. 1997 Oct. 14;94(21):11456-60). Failed termination on a gene can impair the expressionof downstream genes by reducing the accessibility of the promoter to PolII (Greger IH. et al., 2000 “Balancing transcriptional interference andinitiation on the GAL7 promoter of Saccharomyces cerevisiae.” Proc NatlAcad Sci USA. 2000 Jul. 18; 97(15):8415-20). This process, known astranscriptional interference, is particularly relevant in lowereukaryotes, as they often have closely spaced genes.

Termination sequences can also affect the expression of the genes towhich the sequences belong. For example, studies show that inefficienttranscriptional termination in eukaryotes results in an accumulation ofunspliced pre-mRNA (see West, S., and Proudfoot, N.J., 2009“Transcriptional Termination Enhances Protein Expression in Human Cells”Mol Cell. 2009 Feb. 13; 33(3-9); 354-364). Other studies have also shownthat 3′ end processing, can be delayed by inefficient termination (West,S et al., 2008 “Molecular dissection of mammalian RNA polymerase IItranscriptional termination.” Mol Cell. 2008 Mar. 14; 29(5):600-10).Transcriptional termination can also affect mRNA stability by releasingtranscripts from sites of synthesis.

Termination of Transcription Mechanism in Eukaryotes

Transcriptional termination in eukaryotes operates through terminatorsignals that are recognized by protein factors associated with the RNApolymerase II. In some embodiments, the cleavage and polyadenylationspecificity factor (CPSF) and cleavage stimulation factor (CstF)transfer from the carboxyl terminal domain of RNA polymerase II to thepoly-A signal. In some embodiments, the CPSF and CstF factors alsorecruit other proteins to the termination site, which then cleave thetranscript and free the mRNA from the transcription complex. Terminationalso triggers polyadenylation of mRNA transcripts. Illustrative examplesof validated eukaryotic termination factors, and their conservedstructures are discussed in later portions of this document.

Terminator sequences or signals can be operably linked to the 3′ terminiof sequences to be expressed. A variety of known fungal terminators arelikely to be functional in the host strains of the disclosure. Examplesare the A. nidulans trpC terminator, A. niger alpha-glucosidaseterminator, A. niger glucoamylase terminator, Mucor miehei carboxylprotease terminator (see U.S. Pat. No. 5,578,463), Chrysosporiumterminator sequences, e.g. the EG6 terminator, and the Trichodermareesei cellobiohydrolase terminator. In one embodiment, the terminatorsequences are direct repeats (DRs). In one embodiment, the terminatorsequence is the native A. niger pyrG terminator. The native A. nigerpyrG sequence can have the sequence of SEQ ID NO. 5

Terminator Swapping (STOP Swap)

In some embodiments, the present disclosure teaches methods of selectingtermination sequences (“terminators”) with optimal expression propertiesto produce beneficial effects on overall-host strain productivity.

For example, in some embodiments, the present disclosure teaches methodsof identifying one or more terminators and/or generating variants of oneor more terminators within a host cell, which exhibit a range ofexpression strengths (e.g. terminator ladders discussed infra). Aparticular combination of these identified and/or generated terminatorscan be grouped together as a terminator ladder, which is explained inmore detail below.

The terminator ladder in question is then associated with a given geneof interest. Thus, if one has terminators T₁-T₈ (representing eightterminators that have been identified and/or generated to exhibit arange of expression strengths when combined with one or more promoters)and associates the terminator ladder with a single gene of interest in ahost cell (i.e. genetically engineer a host cell with a given terminatoroperably linked to the 3′ end of to a given target gene), then theeffect of each combination of the terminators can be ascertained bycharacterizing each of the engineered strains resulting from eachcombinatorial effort, given that the engineered host cells have anotherwise identical genetic background except the particularterminator(s) associated with the target gene. The resultant host cellsthat are engineered via this process form HTP genetic design libraries.

The HTP genetic design library can refer to the actual physicalmicrobial strain collection that is formed via this process, with eachmember strain being representative of a given terminator operably linkedto a particular target gene, in an otherwise identical geneticbackground, said library being termed a “terminator swap microbialstrain library” or “STOP swap microbial strain library.” In the specificcontext of filamentous fungus (e.g., A. niger), the library can betermed a “terminator swap filamentous fungal strain library,”“terminator swap filamentous A. niger library”, “STOP swap filamentousfungal strain library,” or “STOP swap A. niger strain library,” but theterms can be used synonymously, as filamentous fungus or A. niger arespecific examples of a microbe.

Furthermore, the HTP genetic design library can refer to the collectionof genetic perturbations—in this case a given terminator x operablylinked to a given gene y—said collection being termed a “terminator swaplibrary” or “STOP swap library.”

Further, one can utilize the same terminator ladder comprisingterminators T₁-T₈ to engineer microbes, wherein each of the eightterminators is operably linked to 10 different gene targets. The resultof this procedure would be 80 host cell strains that are otherwiseassumed genetically identical, except for the particular terminatorsoperably linked to a target gene of interest. These 80 host cell strainscould be appropriately screened and characterized and give rise toanother HTP genetic design library. The characterization of themicrobial strains in the HTP genetic design library produces informationand data that can be stored in any database, including withoutlimitation, a relational database, an object-oriented database or ahighly distributed NoSQL database. This data/information could include,for example, a given terminators' (e.g., T₁-T₈) effect when operablylinked to a given gene target. This data/information can also be thebroader set of combinatorial effects that result from operably linkingtwo or more of promoters T₁-T₈ to a given gene target.

The aforementioned examples of eight terminators and 10 target genes ismerely illustrative, as the concept can be applied with any given numberof promoters that have been grouped together based upon exhibition of arange of expression strengths and any given number of target genes.

In summary, utilizing various terminators to modulate expression ofvarious genes in an organism is a powerful tool to optimize a trait ofinterest. The molecular tool of terminator swapping, developed by theinventors, uses a ladder of terminator sequences that have beendemonstrated to vary expression of at least one locus under at least onecondition. This ladder is then systematically applied to a group ofgenes in the organism using high-throughput genome engineering. Thisgroup of genes is determined to have a high likelihood of impacting thetrait of interest based on any one of a number of methods. These couldinclude selection based on known function, or impact on the trait ofinterest, or algorithmic selection based on previously determinedbeneficial genetic diversity.

The resultant HTP genetic design microbial library of organismscontaining a terminator sequence linked to a gene is then assessed forperformance in a high-throughput screening model, and promoter-genelinkages which lead to increased performance are determined and theinformation stored in a database. The collection of geneticperturbations (i.e. given terminator x linked to a given gene y) form a“terminator swap library,” which can be utilized as a source ofpotential genetic alterations to be utilized in microbial engineeringprocessing. Over time, as a greater set of genetic perturbations isimplemented against a greater diversity of microbial backgrounds, eachlibrary becomes more powerful as a corpus of experimentally confirmeddata that can be used to more precisely and predictably design targetedchanges against any background of interest. That is in some embodiments,the present disclosures teaches introduction of one or more geneticchanges into a host cell based on previous experimental results embeddedwithin the meta data associated with any of the genetic design librariesof the disclosure.

Thus, in particular embodiments, terminator swapping is a multi-stepprocess comprising:

1. Selecting a set of “x” terminators to act as a “ladder.” Ideallythese terminators have been shown to lead to highly variable expressionacross multiple genomic loci, but the only requirement is that theyperturb gene expression in some way.

2. Selecting a set of “n” genes to target. This set can be every ORF ina genome, or a subset of ORFs. The subset can be chosen usingannotations on ORFs related to function, by relation to previouslydemonstrated beneficial perturbations (previous promoter swaps, STOPswaps, or SNP swaps), by algorithmic selection based on epistaticinteractions between previously generated perturbations, other selectioncriteria based on hypotheses regarding beneficial ORF to target, orthrough random selection. In other embodiments, the “n” targeted genescan comprise non-protein coding genes, including non-coding RNAs.

3. High-throughput strain engineering to rapidly and in parallel carryout the following genetic modifications: When a native terminator existsat the 3′ end of target gene n and its sequence is known, replace thenative terminator with each of the x terminators in the ladder. When thenative terminator does not exist, or its sequence is unknown, inserteach of the x terminators in the ladder after the gene stop codon.

In this way a “library” (also referred to as a HTP genetic designlibrary) of strains is constructed, wherein each member of the libraryis an instance of x terminator linked to n target, in an otherwiseidentical genetic context. As previously described, combinations ofterminators can be inserted, extending the range of combinatorialpossibilities upon which the library is constructed.

4. High-throughput screening of the library of strains in a contextwhere their performance against one or more metrics is indicative of theperformance that is being optimized.

This foundational process can be extended to provide furtherimprovements in strain performance by, inter alia: (1) Consolidatingmultiple beneficial perturbations into a single strain background,either one at a time in an interactive process, or as multiple changesin a single step. Multiple perturbations can be either a specific set ofdefined changes or a partly randomized, combinatorial library ofchanges. For example, if the set of targets is every gene in a pathway,then sequential regeneration of the library of perturbations into animproved member or members of the previous library of strains canoptimize the expression level of each gene in a pathway regardless ofwhich genes are rate limiting at any given iteration; (2) Feeding theperformance data resulting from the individual and combinatorialgeneration of the library into an algorithm that uses that data topredict an optimum set of perturbations based on the interaction of eachperturbation; and (3) Implementing a combination of the above twoapproaches.

The approach is exemplified in the present disclosure with industrialmicroorganisms, but is applicable to any organism where desired traitscan be identified in a population of genetic mutants. For example, thiscould be used for improving the performance of CHO cells, yeast, insectcells, algae, as well as multi-cellular organisms, such as plants.

5. Sequence Optimization: A Molecular Tool for the Derivation ofOptimized Sequence Microbial Strain Libraries

In one embodiment, the methods of the disclosure comprise codonoptimizing one or more genes expressed by the host organism. Methods foroptimizing codons to improve expression in various hosts are known inthe art and are described in the literature (see U.S. Pat. App. Pub. No.2007/0292918, incorporated herein by reference in its entirety).Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (see also, Murray el al. (1989) Nucl.Acids Res. 17:477-508) can be prepared, for example, to increase therate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence.

Protein expression is governed by a host of factors including those thataffect transcription, mRNA processing, and stability and initiation oftranslation. Optimization can thus address any of a number of sequencefeatures of any particular gene. As a specific example, a rare codoninduced translational pause can result in reduced protein expression. Arare codon induced translational pause includes the presence of codonsin the polynucleotide of interest that are rarely used in the hostorganism may have a negative effect on protein translation due to theirscarcity in the available tRNA pool.

Alternate translational initiation also can result in reducedheterologous protein expression. Alternate translational initiation caninclude a synthetic polynucleotide sequence inadvertently containingmotifs capable of functioning as a ribosome binding site (RBS). Thesesites can result in initiating translation of a truncated protein from agene-internal site. One method of reducing the possibility of producinga truncated protein, which can be difficult to remove duringpurification, includes eliminating putative internal RBS sequences froman optimized polynucleotide sequence.

Repeat-induced polymerase slippage can result in reduced heterologousprotein expression. Repeat-induced polymerase slippage involvesnucleotide sequence repeats that have been shown to cause slippage orstuttering of DNA polymerase which can result in frameshift mutations.Such repeats can also cause slippage of RNA polymerase. In an organismwith a high G+C content bias, there can be a higher degree of repeatscomposed of G or C nucleotide repeats. Therefore, one method of reducingthe possibility of inducing RNA polymerase slippage, includes alteringextended repeats of G or C nucleotides.

Interfering secondary structures also can result in reduced heterologousprotein expression. Secondary structures can sequester the RBS sequenceor initiation codon and have been correlated to a reduction in proteinexpression. Stemloop structures can also be involved in transcriptionalpausing and attenuation. An optimized polynucleotide sequence cancontain minimal secondary structures in the RBS and gene coding regionsof the nucleotide sequence to allow for improved transcription andtranslation.

For example, the optimization process can begin by identifying thedesired amino acid sequence to be expressed by the host. From the aminoacid sequence a candidate polynucleotide or DNA sequence can bedesigned. During the design of the synthetic DNA sequence, the frequencyof codon usage can be compared to the codon usage of the host expressionorganism and rare host codons can be removed from the syntheticsequence. Additionally, the synthetic candidate DNA sequence can bemodified in order to remove undesirable enzyme restriction sites and addor remove any desired signal sequences, linkers or untranslated regions.The synthetic DNA sequence can be analyzed for the presence of secondarystructure that may interfere with the translation process, such as G/Crepeats and stem-loop structures.

6. Epistasis Mapping—A Predictive Analytical Tool Enabling BeneficialGenetic Consolidations

In some embodiments, the present disclosure teaches epistasis mappingmethods for predicting and combining beneficial genetic alterations intoa host cell. The genetic alterations may be created by any of theaforementioned HTP molecular tool sets (e.g., promoter swaps, SNP swaps,start/stop codon exchanges, sequence optimization, and STOP swaps) andthe effect of those genetic alterations would be known from thecharacterization of the derived HTP genetic design microbial strainlibraries. Thus, as used herein, the term epistasis mapping includesmethods of identifying combinations of genetic alterations (e.g.,beneficial SNPs or beneficial promoter/target gene associations) thatare likely to yield increases in host performance.

In embodiments, the epistasis mapping methods of the present disclosureare based on the idea that the combination of beneficial mutations fromtwo different functional groups is more likely to improve hostperformance, as compared to a combination of mutations from the samefunctional group. See, e.g., Costanzo, The Genetic Landscape of a Cell,Science, Vol. 327, Issue 5964, Jan. 22, 2010, pp. 425-431 (incorporatedby reference herein in its entirety).

Mutations from the same functional group are more likely to operate bythe same mechanism, and are thus more likely to exhibit negative orneutral epistasis on overall host performance. In contrast, mutationsfrom different functional groups are more likely to operate byindependent mechanisms, which can lead to improved host performance andin some instances synergistic effects.

Thus, in some embodiments, the present disclosure teaches methods ofanalyzing SNP mutations to identify SNPs predicted to belong todifferent functional groups. In some embodiments, SNP functional groupsimilarity is determined by computing the cosine similarity of mutationinteraction profiles. The present disclosure also illustrates comparingSNPs via a mutation similarity matrix or dendrogram.

Thus, the epistasis mapping procedure provides a method for groupingand/or ranking a diversity of genetic mutations applied in one or moregenetic backgrounds for the purposes of efficient and effectiveconsolidations of said mutations into one or more genetic backgrounds.

In aspects, consolidation is performed with the objective of creatingnovel strains which are optimized for the production of targetbiomolecules. Through the taught epistasis mapping procedure, it ispossible to identify functional groupings of mutations, and suchfunctional groupings enable a consolidation strategy that minimizesundesirable epistatic effects.

As previously explained, the optimization of microbes for use inindustrial fermentation is an important and difficult problem, withbroad implications for the economy, society, and the natural world.Traditionally, microbial engineering has been performed through a slowand uncertain process of random mutagenesis. Such approaches leveragethe natural evolutionary capacity of cells to adapt to artificiallyimposed selection pressure. Such approaches are also limited by therarity of beneficial mutations, the ruggedness of the underlying fitnesslandscape, and more generally underutilize the state of the art incellular and molecular biology.

Modern approaches leverage new understanding of cellular function at themechanistic level and new molecular biology tools to perform targetedgenetic manipulations to specific phenotypic ends. In practice, suchrational approaches are confounded by the underlying complexity ofbiology. Causal mechanisms are poorly understood, particularly whenattempting to combine two or more changes that each has an observedbeneficial effect. Sometimes such consolidations of genetic changesyield positive outcomes (measured by increases in desired phenotypicactivity), although the net positive outcome may be lower than expectedand in some cases higher than expected. In other instances, suchcombinations produce either net neutral effect or a net negative effect.This phenomenon is referred to as epistasis, and is one of thefundamental challenges to microbial engineering (and genetic engineeringgenerally).

As aforementioned, the present HTP genomic engineering platform solvesmany of the problems associated with traditional microbial engineeringapproaches. The present HTP platform uses automation technologies toperform hundreds or thousands of genetic mutations at once. Inparticular aspects, unlike the rational approaches described above, thedisclosed HTP platform enables the parallel construction of thousands ofmutants to more effectively explore large subsets of the relevantgenomic space, as disclosed in U.S. application Ser. No. 15/140,296,entitled Microbial Strain Design System And Methods For ImprovedLarge-Scale Production Of Engineered Nucleotide Sequences, incorporatedby reference herein in its entirety. By trying “everything,” the presentHTP platform sidesteps the difficulties induced by our limitedbiological understanding.

However, at the same time, the present HTP platform faces the problem ofbeing fundamentally limited by the combinatorial explosive size ofgenomic space, and the effectiveness of computational techniques tointerpret the generated data sets given the complexity of geneticinteractions. Techniques are needed to explore subsets of vastcombinatorial spaces in ways that maximize non-random selection ofcombinations that yield desired outcomes.

Somewhat similar HTP approaches have proved effective in the case ofenzyme optimization. In this niche problem, a genomic sequence ofinterest (on the order of 1000 bases), encodes a protein chain with somecomplicated physical configuration. The precise configuration isdetermined by the collective electromagnetic interactions between itsconstituent atomic components. This combination of short genomicsequence and physically constrained folding problem lends itselfspecifically to greedy optimization strategies. That is, it is possibleto individually mutate the sequence at every residue and shuffle theresulting mutants to effectively sample local sequence space at aresolution compatible with the Sequence Activity Response modeling.

However, for full genomic optimizations for biomolecules, suchresidue-centric approaches are insufficient for some important reasons.First, because of the exponential increase in relevant sequence spaceassociated with genomic optimizations for biomolecules. Second, becauseof the added complexity of regulation, expression, and metabolicinteractions in biomolecule synthesis. The present inventors have solvedthese problems via the taught epistasis mapping procedure.

The taught method for modeling epistatic interactions, between acollection of mutations for the purposes of more efficient and effectiveconsolidation of said mutations into one or more genetic backgrounds, isgroundbreaking and highly needed in the art.

When describing the epistasis mapping procedure, the terms “moreefficient” and “more effective” refers to the avoidance of undesirableepistatic interactions among consolidation strains with respect toparticular phenotypic objectives.

As the process has been generally elaborated upon above, a more specificworkflow example will now be described.

First, one begins with a library of M mutations and one or more geneticbackgrounds (e.g., parent filamentous fungal strains). Neither thechoice of library nor the choice of genetic backgrounds is specific tothe method described here. But in a particular implementation, a libraryof mutations may include exclusively, or in combination: SNP swaplibraries, Promoter swap libraries, or any other mutation librarydescribed herein.

In one implementation, only a single genetic background is provided. Inthis case, a collection of distinct genetic backgrounds (microbialmutants) will first be generated from this single background. This maybe achieved by applying the primary library of mutations (or some subsetthereof) to the given background for example, application of a HTPgenetic design library of particular SNPs or a HTP genetic designlibrary of particular promoters to the given genetic background, tocreate a population (perhaps 100's or 1,000's) of microbial mutants withan identical genetic background except for the particular geneticalteration from the given HTP genetic design library incorporatedtherein. As detailed below, this embodiment can lead to a combinatoriallibrary or pairwise library.

In another implementation, a collection of distinct known geneticbackgrounds may simply be given. As detailed below, this embodiment canlead to a subset of a combinatorial library.

In a particular implementation, the number of genetic backgrounds andgenetic diversity between these backgrounds (measured in number ofmutations or sequence edit distance or the like) is determined tomaximize the effectiveness of this method.

A genetic background may be a natural, native or wild-type strain or amutated, engineered strain. N distinct background strains may berepresented by a vector b. In one example, the background b mayrepresent engineered backgrounds formed by applying N primary mutationsm₀=(m₁, m₂, . . . m_(N)) to a wild-type background strain b₀ to form theN mutated background strains b=m₀ b₀=(m₁b₀, m₂b₀, . . . m_(N) b₀), wherem_(i)b₀ represents the application of mutation m_(i) to backgroundstrain b₀.

In either case (i.e. a single provided genetic background or acollection of genetic backgrounds), the result is a collection of Ngenetically distinct backgrounds. Relevant phenotypes are measured foreach background.

Second, each mutation in a collection of M mutations m₁ is applied toeach background within the collection of N background strains b to forma collection of M×N mutants. In the implementation where the Nbackgrounds were themselves obtained by applying the primary set ofmutations m₀ (as described above), the resulting set of mutants willsometimes be referred to as a combinatorial library or a pairwiselibrary. In another implementation, in which a collection of knownbackgrounds has been provided explicitly, the resulting set of mutantsmay be referred to as a subset of a combinatorial library. Similar togeneration of engineered background vectors, in embodiments, the inputinterface 202 (see, FIG. 14) receives the mutation vector m₁ and thebackground vector b, and a specified operation such as cross product.

Continuing with the engineered background example above, forming the M×Ncombinatorial library may be represented by the matrix formed by m₁×m₀b₀, the cross product of m₁ applied to the N backgrounds of b=m₀ b₀,where each mutation in m₁ is applied to each background strain within b.Each ith row of the resulting M×N matrix represents the application ofthe ith mutation within m₁ to all the strains within backgroundcollection b. In one embodiment, m₁=m₀ and the matrix represents thepairwise application of the same mutations to starting strain b₀. Inthat case, the matrix is symmetric about its diagonal (M=N), and thediagonal may be ignored in any analysis since it represents theapplication of the same mutation twice.

In embodiments, forming the M×N matrix may be achieved by inputting intothe input interface 202 (see, FIG. 14) the compound expression m₁×m₀b₀.The component vectors of the expression may be input directly with theirelements explicitly specified, via one or more DNA specifications, or ascalls to the library 206 to enable retrieval of the vectors duringinterpretation by interpreter 204. As described in U.S. patentapplication Ser. No. 15/140,296, entitled “Microbial Strain DesignSystem and Methods for Improved Large Scale Production of EngineeredNucleotide Sequences,” via the interpreter 204, execution engine 207,order placement engine 208, and factory 210, the LIMS system 200generates the microbial strains specified by the input expression.

Third, with reference to FIG. 19, the analysis equipment 214 (see, FIG.14) measures phenotypic responses for each mutant within the M×Ncombinatorial library matrix (4202). As such, the collection ofresponses can be construed as an M×N Response Matrix R. Each element ofR may be represented as r_(ij)=y(m_(i), m_(j)), where y represents theresponse (performance) of background strain b_(j) within engineeredcollection b as mutated by mutation m_(i). For simplicity, andpracticality, we assume pairwise mutations where m₁=m₀. Where, as here,the set of mutations represents a pairwise mutation library, theresulting matrix may also be referred to as a gene interaction matrixor, more particularly, as a mutation interaction matrix.

Those skilled in the art will recognize that, in some embodiments,operations related to epistatic effects and predictive strain design maybe performed entirely through automated means of the LIMS system 200,e.g., by the analysis equipment 214 (see, FIG. 14), or by humanimplementation, or through a combination of automated and manual means.When an operation is not fully automated, the elements of the LIMSsystem 200, e.g., analysis equipment 214, may, for example, receive theresults of the human performance of the operations rather than generateresults through its own operational capabilities. As described elsewhereherein, components of the LIMS system 200, such as the analysisequipment 214, may be implemented wholly or partially by one or morecomputer systems. In some embodiments, in particular where operationsrelated to predictive strain design are performed by a combination ofautomated and manual means, the analysis equipment 214 may include notonly computer hardware, software or firmware (or a combination thereof),but also equipment operated by a human operator such as that listed inTable 3 below, e.g., the equipment listed under the category of“Evaluate performance.”

Fourth, the analysis equipment 214 (see, FIG. 14) normalizes theresponse matrix. Normalization consists of a manual and/or, in thisembodiment, automated processes of adjusting measured response valuesfor the purpose of removing bias and/or isolating the relevant portionsof the effect specific to this method. With respect to FIG. 19, thefirst step 4202 may include obtaining normalized measured data. Ingeneral, in the claims directed to predictive strain design andepistasis mapping, the terms “performance measure” or “measuredperformance” or the like may be used to describe a metric that reflectsmeasured data, whether raw or processed in some manner, e.g., normalizeddata. In a particular implementation, normalization may be performed bysubtracting a previously measured background response from the measuredresponse value. In that implementation, the resulting response elementsmay be formed as r_(ij)=y(m_(i), m_(j))−y(m_(j)), where y(m_(j)) is theresponse of the engineered background strain b_(j) within engineeredcollection b caused by application of primary mutation m_(j) to parentstrain b₀. Note that each row of the normalized response matrix istreated as a response profile for its corresponding mutation. That is,the ith row describes the relative effect of the corresponding mutationmi applied to all the background strains b_(j) for j=1 to N.

With respect to the example of pairwise mutations, the combinedperformance/response of strains resulting from two mutations may begreater than, less than, or equal to the performance/response of thestrain to each of the mutations individually. This effect is known as“epistasis,” and may, in some embodiments, be represented ase_(ij)=y(m_(i), m_(j))−(y(m_(i))+y(m_(j))). Variations of thismathematical representation are possible, and may depend upon, forexample, how the individual changes biologically interact. As notedabove, mutations from the same functional group are more likely tooperate by the same mechanism, and are thus more likely to exhibitnegative or neutral epistasis on overall host performance. In contrast,mutations from different functional groups are more likely to operate byindependent mechanisms, which can lead to improved host performance byreducing redundant mutative effects, for example. Thus, mutations thatyield dissimilar responses are more likely to combine in an additivemanner than mutations that yield similar responses. This leads to thecomputation of similarity in the next step.

Fifth, the analysis equipment 214 measures the similarity among theresponses—in the pairwise mutation example, the similarity between theeffects of the ith mutation and jth (e.g., primary) mutation within theresponse matrix (4204). Recall that the ith row of R represents theperformance effects of the ith mutation m_(i) on the N backgroundstrains, each of which may be itself the result of engineered mutationsas described above. Thus, the similarity between the effects of the ithand jth mutations may be represented by the similarity s_(ij) betweenthe ith and jth rows, ρ_(i) and ρ_(j), respectively, to form asimilarity matrix S. Similarity may be measured using many knowntechniques, such as cross-correlation or absolute cosine similarity,e.g., s_(ij)=abs(cos(ρ_(i), ρ_(j))).

As an alternative or supplement to a metric like cosine similarity,response profiles may be clustered to determine degree of similarity.Clustering may be performed by use of a distance-based clusteringalgorithms (e.g. k-mean, hierarchical agglomerative, etc.) inconjunction with suitable distance measure (e.g. Euclidean, Hamming,etc). Alternatively, clustering may be performed using similarity basedclustering algorithms (e.g. spectral, min-cut, etc.) with a suitablesimilarity measure (e.g. cosine, correlation, etc). Of course, distancemeasures may be mapped to similarity measures and vice-versa via anynumber of standard functional operations (e.g., the exponentialfunction). In one implementation, hierarchical agglomerative clusteringmay be used in conjunction absolute cosine similarity.

As an example of clustering, let C be a clustering of mutations m_(i)into k distinct clusters. Let C be the cluster membership matrix, wherec_(ij) is the degree to which mutation i belongs to cluster j, a valuebetween 0 and 1. The cluster-based similarity between mutations i and jis then given by C_(i)×C_(j) (the dot product of the ith and jth rows ofC). In general, the cluster-based similarity matrix is given by CC^(T)(that is, C times C-transpose). In the case of hard-clustering (amutation belongs to exactly one cluster), the similarity between twomutations is 1 if they belong to the same cluster and 0 if not.

As is described in Costanzo, The Genetic Landscape of a Cell, Science,Vol. 327, Issue 5964, Jan. 22, 2010, pp. 425-431 (incorporated byreference herein in its entirety), such a clustering of mutationresponse profiles relates to an approximate mapping of a cell'sunderlying functional organization. That is, mutations that clustertogether tend to be related by an underlying biological process ormetabolic pathway. Such mutations are referred to herein as a“functional group.” The key observation of this method is that if twomutations operate by the same biological process or pathway, thenobserved effects (and notably observed benefits) may be redundant.Conversely, if two mutations operate by distant mechanism, then it isless likely that beneficial effects will be redundant.

Sixth, based on the epistatic effect, the analysis equipment 214 selectspairs of mutations that lead to dissimilar responses, e.g., their cosinesimilarity metric falls below a similarity threshold, or their responsesfall within sufficiently separated clusters, as shown in FIG. 19 (4206).Based on their dissimilarity, the selected pairs of mutations shouldconsolidate into background strains better than similar pairs.

Based upon the selected pairs of mutations that lead to sufficientlydissimilar responses, the LIMS system (e.g., all of or some combinationof interpreter 204, execution engine 207, order placer 208, and factory210) may be used to design microbial strains having those selectedmutations (4208). In embodiments, as described below and elsewhereherein, epistatic effects may be built into, or used in conjunction withthe predictive model to weight or filter strain selection.

It is assumed that it is possible to estimate the performance (a.k.a.score) of a hypothetical strain obtained by consolidating a collectionof mutations from the library into a particular background via somepreferred predictive model. A representative predictive model utilizedin the taught methods is provided in the below section entitled“Predictive Strain Design” that is found in the larger section of:“Computational Analysis and Prediction of Effects of Genome-Wide GeneticDesign Criteria.”

When employing a predictive strain design technique such as linearregression, the analysis equipment 214 may restrict the model tomutations having low similarity measures by, e.g., filtering theregression results to keep only sufficiently dissimilar mutations.Alternatively, the predictive model may be weighted with the similaritymatrix. For example, some embodiments may employ a weighted leastsquares regression using the similarity matrix to characterize theinterdependencies of the proposed mutations. As an example, weightingmay be performed by applying the “kernel” trick to the regression model.(To the extent that the “kernel trick” is general to many machinelearning modeling approaches, this re-weighting strategy is notrestricted to linear regression.)

Such methods are known to one skilled in the art. In embodiments, thekernel is a matrix having elements 1−w*s_(ij) where 1 is an element ofthe identity matrix, and w is a real value between 0 and 1. When w=0,this reduces to a standard regression model. In practice, the value of wwill be tied to the accuracy (r² value or root mean square error (RMSE))of the predictive model when evaluated against the pairwisecombinatorial constructs and their associate effects y(m_(i), m_(j)). Inone simple implementation, w is defined as w=1−r². In this case, whenthe model is fully predictive, w=1−r²=0 and consolidation is basedsolely on the predictive model and epistatic mapping procedure plays norole. On the other hand, when the predictive model is not predictive atall, w=1−r²=1 and consolidation is based solely on the epistatic mappingprocedure. During each iteration, the accuracy can be assessed todetermine whether model performance is improving.

It should be clear that the epistatic mapping procedure described hereindoes not depend on which model is used by the analysis equipment 214.Given such a predictive model, it is possible to score and rank allhypothetical strains accessible to the mutation library viacombinatorial consolidation.

In some embodiments, to account for epistatic effects, the dissimilarmutation response profiles may be used by the analysis equipment 214 toaugment the score and rank associated with each hypothetical strain fromthe predictive model. This procedure may be thought of broadly as are-weighting of scores, so as to favor candidate strains with dissimilarresponse profiles (e.g., strains drawn from a diversity of clusters). Inone simple implementation, a strain may have its score reduced by thenumber of constituent mutations that do not satisfy the dissimilaritythreshold or that are drawn from the same cluster (with suitableweighting). In a particular implementation, a hypothetical strain'sperformance estimate may be reduced by the sum of terms in thesimilarity matrix associated with all pairs of constituent mutationsassociated with the hypothetical strain (again with suitable weighting).Hypothetical strains may be re-ranked using these augmented scores. Inpractice, such re-weighting calculations may be performed in conjunctionwith the initial scoring estimation.

The result is a collection of hypothetical strains with score and rankaugmented to more effectively avoid confounding epistatic interactions.Hypothetical strains may be constructed at this time, or they may bepassed to another computational method for subsequent analysis or use.

Those skilled in the art will recognize that epistasis mapping anditerative predictive strain design as described herein are not limitedto employing only pairwise mutations, but may be expanded to thesimultaneous application of many more mutations to a background strain.In another embodiment, additional mutations may be applied sequentiallyto strains that have already been mutated using mutations selectedaccording to the predictive methods described herein. In anotherembodiment, epistatic effects are imputed by applying the same geneticmutation to a number of strain backgrounds that differ slightly fromeach other, and noting any significant differences in positive responseprofiles among the modified strain backgrounds.

Genetic Design & Microbial Engineering: Directed Genome Editing withTargeted Nucleases

Metabolic engineering relies heavily on the alteration of key genesinvolved, both directly and indirectly, in the metabolism, regulation,and catabolism of molecules. It is often useful to precisely introducesmall and large changes such as single nucleotide polymorphisms,insertions, or deletions into the genome to alter metabolic pathways.Such changes can also be used to introduce, delete or replace largerregions of genetic material such as promoters, terminators, genes, oreven gene clusters.

Through sequence homology, these crRNAs guide a Cas nuclease to thespecified exogenous genetic material, which must also contain anuclease-specific sequence known as a protospacer adjacent motif (PAM).The CRISPR complex binds to the foreign DNA and cleaves it.

In one aspect provided herein, a host or parental strain of fungi (e.g.,filamentous fungi as provided herein) that contains a metabolic pathwayof interest that produces a molecule or biologic of interest can bemodified by CRISPR. In one embodiment, a protoplast capable of beingtransformed is generated from the host or parental strain using theprotoplasting methods provided herein and is transformed with aribonucleoprotein complex (RNP-complex or CRISPR RNP). The RNP-complexcomprises a nucleic acid guided nuclease as provided herein (e.g., Cas9)that is complexed with a guide nucleic acid as provided herein (e.g.,guide RNA (gRNA)). When guided by an RNA, the nucleic acid guidednuclease can be referred to herein as an RNA guided nuclease or an RNAguided endonuclease. The guide nucleic acid (e.g., gRNA) can comprise atargeting segment that is a guide sequence that is complementary to aportion of a target gene or nucleic acid sequence present in the host orparental strain of fungi (e.g., filamentous fungi as provided herein).In one embodiment, a protoplast can be transformed with 2 or moreRNP-complexes such that each RNP-complex comprises a nucleic acid guidedendonuclease (e.g., Cas9) complexed with a guide nucleic acid (e.g.,gRNA). In some cases, each guide nucleic acid (e.g., gRNA) in the 2 ormore RNP-complexes can comprise guide sequence complementary to adifferent target gene or nucleic acid sequence. In some cases, eachguide nucleic acid (e.g., gRNA) in the 2 or more RNP-complexes cancomprise guide sequences to the same target gene or nucleic acidsequence. In cases where there are 3 or more RNP-complexes, there can bea subset of the RNP-complexes that can comprise guide sequencescomplementary to the same target gene or nucleic acid sequence and asubset or subsets of the RNP-complexes that can comprise guide sequencecomplementary to a different target gene or nucleic acid. In cases where2 or more RNP-complexes are directed to the same target nucleic acidsequence via their respective targeting segment, each of theRNP-complexes can comprise a guide sequence or targeting segment that iscomplementary to a different or separate portion of said target gene ornucleic acid. Further to these embodiments, multiple gRNAs targetingmultiple loci can be expressed in the same cell or organism (multiplexexpression of gRNAs). Pooled gRNA libraries can be used to identifygenes that are important to a given phenotype. Current libraries areavailable for gene knockout, as well as transcriptional activation orrepression. Combined with the power of next-generation sequencing,CRISPR can be a robust system for genome-wide screening. Each gRNA cancomprise a CRISPR RNA (crRNA) annealed to a transactivating crRNA(tracrRNA) or can comprise a single gRNA (sgRNA) that comprises a singletranscript comprising a crRNA and a tracrRNA.

In one embodiment, the host or parental fungi has a functioning NHEJpathway. Further to this embodiment, transformation of protoplastsderived from the host or parental fungi with a single RNP-complex ormultiple RNP-complexes generates strand break(s) within the targetgene(s) in the genome that are repaired via the NHEJ pathway. Repairusing the NHEJ pathway can lead to indels within the target gene(s). Theindels in the target gene(s) can result in amino acid deletions,insertions, or frameshift mutations leading to premature stop codonswithin the open reading frame (ORF) of the targeted gene(s). In afurther embodiment, the strand breaks within the target gene(s) can berepaired by using homology directed repair (HDR). HDR mediated repaircan be facilitated by co-transforming the protoplasts derived from thehost or parental fungi with a donor DNA sequence. The donor DNA sequencecan comprise a desired genetic perturbation (e.g., deletion, insertion,and/or single nucleotide polymorphism). In this embodiment, the RNPcleaves the target gene specified by the one or more gRNAs. The donorDNA can then be used as a template for the homologous recombinationmachinery to incorporate the desired genetic perturbation formodification of the metabolic pathway or molecule/biologic of interest.The donor DNA can be single-stranded, double-stranded or adouble-stranded plasmid. The donor DNA can lack a PAM sequence orcomprise a scrambled, altered or non-functional PAM in order to preventre-cleavage. In some cases, the donor DNA can contain a functional ornon-altered PAM site (see FIGS. 56A-B). The mutated or edited sequencein the donor DNA (also flanked by the regions of homology) preventsre-cleavage by the RNP after the mutation(s) has/have been incorporatedinto the genome. In some embodiments, the HDR pathway can be favored byperforming the transformations in protoplasts derived from host orparental fungi that do not possess a functioning NHEJ pathway. Disablingthe NHEJ pathway can be achieved using any of the methods providedherein.

In some embodiments, the protoplasts can be co-transformed with the RNPcomplex, the donor DNA and a vector comprising a selectable marker. Thevector can interact with the other components by enabling identificationand/or survival of only transformationally competent protoplasts. Thiscan facilitate identification of transformed and correctly editedstrains. Iterative rounds of editing are possible because the plasmidcan be cured. See FIG. 53.

Further to the above embodiments, the nucleic acid guided nuclease foruse in the methods provided herein can be any of the nucleic acid guidednucleases known in the art and/or provided herein. In one embodiment,the nucleic acid guided nuclease is a Class 2 CRISPR-Cas System nucleicacid guided nuclease. The Class 2 CRISPR-Cas system nucleic acid guidednuclease can be selected from any one or more of the following: Type II,Type IIA, Type IIB, Type IIC, Type V, and Type VI nucleic acid guidednuclease as described herein. The Class 2 CRISPR-Cas system nucleic acidguided nuclease can be any one or more of the following: Cas9, Cas12a,Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c or homologs,orthologs, mutants, variants or modified versions thereof.

Organisms Amenable to Genetic Design

The disclosed HTP genomic engineering platform is exemplified withindustrial microbial cell cultures (e.g., A. niger), but is applicableto any coenocytic host cell organism where desired traits can beidentified in a population of genetic mutants.

Further, as set forth in the introduction, the current disclosureprovides for a HTP genomic engineering platform to improve host cellcharacteristics in filamentous fungal systems and solves many problemsthat have previously prevented the development of such a systeminfilamentous fungus

Thus, as used herein, the term “microorganism” should be taken broadly.It includes, but is not limited to, the two prokaryotic domains,Bacteria and Archaea, as well as certain eukaryotic fungi and protists.However, in certain aspects, “higher” eukaryotic organisms such asinsects, plants, and animals can be utilized in the methods taughtherein.

Suitable host cells include, but are not limited to: bacterial cells,algal cells, plant cells, fungal cells, insect cells, and mammaliancells. In one illustrative embodiment, suitable host cells include A.niger.

In one embodiment, the methods and systems provided herein use fungalelements derived from filamentous fungus that are capable of beingreadily separated from other such elements in a culture medium, and arecapable of reproducing itself. For example, the fungal elements can be aspore, propagule, hyphal fragment, protoplast or micropellet. In apreferred embodiment, the systems and methods provided herein utilizeprotoplasts derived from filamentous fungus. Suitable filamentous fungihost cells include, for example, any filamentous forms of the divisionAscomycota, Deuteromycota, Zygomycota or Fungi imperfecti. Suitablefilamentous fungi host cells include, for example, any filamentous formsof the subdivision Eumycotina. (see, e.g., Hawksworth et al., InAinsworth and Bisby's Dictionary of The Fungi, 8^(th) edition, 1995, CABInternational, University Press, Cambridge, UK, which is incorporatedherein by reference). In certain illustrative, but non-limitingembodiments, the filamentous fungal host cell may be a cell of a speciesof: Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Filibasidium, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea,Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora,Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor,Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces,Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma,Verticillium, Volvariella, or teleomorphs, or anamorphs, and synonyms ortaxonomic equivalents thereof. In one embodiment, the filamentous fungusis selected from the group consisting of A. nidulans, A. oryzae, A.sojae, and Aspergilli of the A. niger Group. In a preferred embodiment,the filamentous fungus is Aspergillus niger.

In one embodiment, the filamentous fungus is a production strainselected from Aspergillus foetidus ACM 3996 (=FRR 3558), Magnaporthegrisea Guy-II or Phanerochaete chrysosporium RP78. In a separateembodiment, the filamentous fungus is an A. niger production strainknown in the art. Examples of A. niger production strains for use in themethods provided herein can include A. niger ATCC 11414, ATCC 1015, ACM4992 (=ATCC 9142), ACM 4993 (=ATCC 10577), ACM 4994 (=ATCC 12846),ATCC26550, ATCC 11414, N402, CBS 513.88 or NRRL3 (ATCC 9029, CBS120.49).

In another embodiment of the present disclosure, specific mutants of thefungal species are used for the methods and systems provided herein. Inone embodiment, specific mutants of the fungal species are used whichare suitable for the high-throughput and/or automated methods andsystems provided herein. Examples of such mutants can be strains thatprotoplast very well; strains that produce mainly or, more preferably,only protoplasts with a single nucleus; strains that regenerateefficiently in microtiter plates, strains that regenerate faster and/orstrains that take up polynucleotide (e.g., DNA) molecules efficiently,strains that produce cultures of low viscosity such as, for example,cells that produce hyphae in culture that are not so entangled as toprevent isolation of single clones and/or raise the viscosity of theculture, strains that have reduced random integration (e.g., disablednon-homologous end joining pathway) or combinations thereof. In yetanother embodiment, a specific mutant strain for use in the methods andsystems provided herein can be strains lacking a selectable marker genesuch as, for example, uridine-requiring mutant strains. These mutantstrains can be either deficient in orotidine 5 phosphate decarboxylase(OMPD) or rotate p-ribosyl transferase (OPRT) encoded by the pyrG orpyrE gene, respectively (T. Goosen et al., Curr Genet. 1987, 11:499 503;J. Begueret et al., Gene. 1984 32:487 92.

In one embodiment, specific mutant strains for use in the methods andsystems provided herein are strains that possess a compact cellularmorphology characterized by shorter hyphae and a more yeast-likeappearance. Examples of such mutants can be filamentous fungal cellswith altered gas1 expression as described in US20140220689.

In still another embodiment, mutant strains for use in the methods andsystems provided herein are modified in their DNA repair system in sucha way that they are extremely efficient in homologous recombinationand/or extremely inefficient in random integration. The efficiency oftargeted integration of a nucleic acid construct into the genome of thehost cell by homologous recombination, i.e. integration in apredetermined target locus, can be increased by augmented homologousrecombination abilities and/or diminished non-homologous recombinationabilities of the host cell. Augmentation of homologous recombination canbe achieved by overexpressing one or more genes involved in homologousrecombination (e.g., Rad51 and/or Rad52 protein). Removal, disruption orreduction in non-homologous recombination or the non-homologous endjoining (NHEJ) pathway in the host cells of the present disclosure canbe achieved by any method known in that art such as, for example, by useof an antibody, a chemical inhibitor, a protein inhibitor, a physicalinhibitor, a peptide inhibitor, or an anti-sense or RNAi moleculedirected against a component of the non-homologous recombination (NHR)or NHEJ pathway (e.g., yeast KU70, yeast KU80 or homologues thereof).Inhibition of the NHEJ pathway can be achieved using chemical inhibitorssuch as described in Arras S M D, Fraser J A (2016), “ChemicalInhibitors of Non-Homologous End Joining Increase Targeted ConstructIntegration in Cryptococcus neoformans” PloS ONE 11 (9): e0163049, thecontents of which are hereby incorporated by reference. Treatment withthe chemical inhibitor(s) to facilitate disabling or reducing the NHEJpathway can be before and/or during generation of protoplasts.Alternatively, a host-cell for use in the methods provided herein can bedeficient in one or more genes (e.g., yeast ku70, ku80 or homologuesthereof) of the NHR pathway. Examples of such mutants are cells with adeficient hdfA or hdfB gene as described in WO 05/95624. Examples ofchemical inhibitors for use in inhibiting NHR in host cells for use inthe methods provided herein can be W-7, chlorpromazine, vanillin,Nu7026, Nu7441, mirin, SCR7, AG14361 or a combination thereof asdescribed in Arras SDM et al (2016) Chemical Inhibitors ofNon-Homologous End Joining Increase Targeted Construct Integration inCryptococcus neoformans. PloS One 11(9).

In one embodiment, a mutant strain of filamentous fungal cell for use inthe methods and systems provided herein have a disabled or reducednon-homologous end-joining (NHEJ) pathway and possess a yeast-like,non-mycelium forming phenotype when grown in culture (e.g., submergedculture).

In another embodiment, filamentous fungal cells for use in the methodsand systems provided herein have a disabled or reduced NHEJ pathway dueto treatment with a chemical inhibitor (e.g., W-7, chlorpromazine,vanillin, Nu7026, Nu7441, mirin, SCR7, AG14361 or any combinationthereof) and possess a yeast-like, non-mycelium forming phenotype whengrown in culture (e.g., submerged culture). In one embodiment, thechemical inhibitor is W-7. As reflected in FIG. 29, a filamentous fungalhost cell (e.g., A. niger) can be treated with a minimum inhibitoryconcentration (MIC) of W-7 that can be host strain dependent.

In some embodiments, the spore, propagule, hyphal fragment, protoplast,or micropellet are isolated to a clonal population. In some embodiments,the spore, propagule, hyphal fragment, protoplast, or micropellet aretransformed prior to isolation. In some embodiments, the spore,propagule, hyphal fragment, protoplast, or micropellet are isolated to aclonal population in a microtiter plate or a microtiter well. Further tothe above embodiments, the clonal populations are derived from a singlespore, propagule, hyphal fragment, protoplast, or micropellet. In someembodiments, the spore, propagule, hyphal fragment, protoplast, ormicropellet is in a diluted solution, and only a single spore,propagule, hyphal fragment, protoplast, or micropellet is transferred toa microtiter plate or well. In some embodiments, the spore, propagule,hyphal fragment, protoplast, or micropellet are diluted to aconcentration to which it can be optically distinguished as a singlespore, propagule, hyphal fragment, protoplast, or micropellet, which isthen singly transferred to a microtiter plate or well. In someembodiments, the spore, propagule, hyphal fragment, protoplast, ormicropellet are diluted using the Poisson distribution where there is astatistical probability of transferring only one spore, propagule,hyphal fragment, protoplast, or micropellet to the microtiter plate orwell. In some embodiments, the spore, propagule, hyphal fragment,protoplast, or micropellet are transferred to any container, including amicrotiter plate or well. In some embodiments, the opticaldistinguishment is performed by CellenONE, Berkeley Lights Beaconinstrument, FACS machine, Cytena, or other like instrument. An exampleof a workflow for isolating a singular spore, propagule, hyphalfragment, protoplast, or micropellet for use in the methods providedherein can be seen in FIG. 54B. Further to any of the above embodiments,the spore, propagule, hyphal fragment, protoplast, or micropellet can befrom any fungal cell provided herein. In one embodiment, the fungal cellis a filamentous fungal cell. The filamentous fungal cell can befilamentous fungal cell provided herein such as, for example, A. niger.

Generating Genetic Diversity Pools for Utilization in the Genetic Design& HTP Microbial Engineering Platform

In some embodiments, the methods of the present disclosure arecharacterized as genetic design. As used herein, the term genetic designrefers to the reconstruction or alteration of a host organism's genomethrough the identification and selection of the most optimum variants ofa particular gene, portion of a gene, promoter, stop codon, 5′UTR,3′UTR, or other DNA sequence to design and create new superior hostcells.

In some embodiments, a first step in the genetic design methods of thepresent disclosure is to obtain an initial genetic diversity poolpopulation with a plurality of sequence variations from which a new hostgenome may be reconstructed.

In some embodiments, a subsequent step in the genetic design methodstaught herein is to use one or more of the aforementioned HTP moleculartool sets (e.g. SNP swapping or promoter swapping) to construct HTPgenetic design libraries, which then function as drivers of the genomicengineering process, by providing libraries of particular genomicalterations for testing in a host cell.

Harnessing Diversity Pools from Existing Wild-Type Strains

In some embodiments, the present disclosure teaches methods foridentifying the sequence diversity present among microbes of a givenwild-type population. Therefore, a diversity pool can be a given numbern of wild-type microbes utilized for analysis, with said microbes'genomes representing the “diversity pool.”

In some embodiments, the diversity pools can be the result of existingdiversity present in the natural genetic variation among said wild-typemicrobes. This variation may result from strain variants of a given hostcell or may be the result of the microbes being different speciesentirely. Genetic variations can include any differences in the geneticsequence of the strains, whether naturally occurring or not. In someembodiments, genetic variations can include SNPs swaps, PRO swaps,Start/Stop Codon swaps, or STOP swaps, among others.

Harnessing Diversity Pools from Existing Industrial Strain Variants

In other embodiments of the present disclosure, diversity pools arestrain variants created during traditional strain improvement processes(e.g., one or more host organism strains generated via random mutationand selected for improved yields over the years). Thus, in someembodiments, the diversity pool or host organisms can comprise acollection of historical production strains.

In particular aspects, a diversity pool may be an original parentmicrobial strain (S₁) with a “baseline” genetic sequence at a particulartime point (S₁Gen₁) and then any number of subsequent offspring strains(S₂, S₃, S₄, S₅, etc., generalizable to S_(2-n)) that werederived/developed from said S₁ strain and that have a different genome(S_(2-n)Gen_(2-n)), in relation to the baseline genome of S₁.

For example, in some embodiments, the present disclosure teachessequencing the microbial genomes in a diversity pool to identify theSNP's present in each strain. In one embodiment, the strains of thediversity pool are historical microbial production strains. Thus, adiversity pool of the present disclosure can include for example, anindustrial base strain, and one or more mutated industrial strainsproduced via traditional strain improvement programs.

Once all SNPs in the diversity pool are identified, the presentdisclosure teaches methods of SNP swapping and screening methods todelineate (i.e. quantify and characterize) the effects (e.g. creation ofa phenotype of interest) of SNPs individually and in groups. Thus, asaforementioned, an initial step in the taught platform can be to obtainan initial genetic diversity pool population with a plurality ofsequence variations, e.g. SNPs. Then, a subsequent step in the taughtplatform can be to use one or more of the aforementioned HTP moleculartool sets (e.g. SNP swapping) to construct HTP genetic design libraries,which then function as drivers of the genomic engineering process, byproviding libraries of particular genomic alterations for testing in amicrobe.

In some embodiments, the SNP swapping methods of the present disclosurecomprise the step of introducing one or more SNPs identified in amutated strain (e.g., a strain from amongst S_(2-n)Gen_(2-n)) to a basestrain (S₁Gen₁) or wild-type strain.

In other embodiments, the SNP swapping methods of the present disclosurecomprise the step of removing one or more SNPs identified in a mutatedstrain (e.g., a strain from amongst S_(2-n)Gen_(2-n)).

Creating Diversity Pools Via Mutagenesis

In some embodiments, the mutations of interest in a given diversity poolpopulation of cells can be artificially generated by any means formutating strains, including mutagenic chemicals, or radiation. The term“mutagenizing” is used herein to refer to a method for inducing one ormore genetic modifications in cellular nucleic acid material.

The term “genetic modification” refers to any alteration of DNA.Representative gene modifications include nucleotide insertions,deletions, substitutions, and combinations thereof, and can be as smallas a single base or as large as tens of thousands of bases. Thus, theterm “genetic modification” encompasses inversions of a nucleotidesequence and other chromosomal rearrangements, whereby the position ororientation of DNA comprising a region of a chromosome is altered. Achromosomal rearrangement can comprise an intrachromosomal rearrangementor an interchromosomal rearrangement.

In one embodiment, the mutagenizing methods employed in the presentlyclaimed subject matter are substantially random such that a geneticmodification can occur at any available nucleotide position within thenucleic acid material to be mutagenized. Stated another way, in oneembodiment, the mutagenizing does not show a preference or increasedfrequency of occurrence at particular nucleotide sequences.

The methods of the disclosure can employ any mutagenic agent including,but not limited to: ultraviolet light, X-ray radiation, gamma radiation,N-ethyl-N-nitrosourea (ENU), methyinitrosourea (MNU), procarbazine(PRC), triethylene melamine (TEM), acrylamide monomer (AA), chlorambucil(CHL), melphalan (MLP), cyclophosphamide (CPP), diethyl sulfate (DES),ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS),6-mercaptopurine (6-MP), mitomycin-C (MMC),N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ³H₂O, and urethane (UR)(See e.g., Rinchik, 1991; Marker el al., 1997; and Russell, 1990).Additional mutagenic agents are well known to persons having skill inthe art, including those described inwww.iephb.nw.ru/˜spirov/hazard/mutagen_1st.html.

The term “mutagenizing” also encompasses a method for altering (e.g., bytargeted mutation) or modulating a cell function, to thereby enhance arate, quality, or extent of mutagenesis. For example, a cell can bealtered or modulated to thereby be dysfunctional or deficient in DNArepair, mutagen metabolism, mutagen sensitivity, genomic stability, orcombinations thereof. Thus, disruption of gene functions that normallymaintain genomic stability can be used to enhance mutagenesis.Representative targets of disruption include, but are not limited to DNAligase I (Bentley et al., 2002) and casein kinase I (U.S. Pat. No.6,060,296).

In some embodiments, site-specific mutagenesis (e.g., primer-directedmutagenesis using a commercially available kit such as the TransformerSite Directed mutagenesis kit (Clontech)) is used to make a plurality ofchanges throughout a nucleic acid sequence in order to generate nucleicacid encoding a cleavage enzyme of the present disclosure.

The frequency of genetic modification upon exposure to one or moremutagenic agents can be modulated by varying dose and/or repetition oftreatment, and can be tailored for a particular application.

Thus, in some embodiments, “mutagenesis” as used herein comprises alltechniques known in the art for inducing mutations, includingerror-prone PCR mutagenesis, oligonucleotide-directed mutagenesis,site-directed mutagenesis, and iterative sequence recombination by anyof the techniques described herein.

Single Locus Mutations to Generate Diversity

In some embodiments, the present disclosure teaches mutating cellpopulations by introducing, deleting, or replacing selected portions ofgenomic DNA. Thus, in some embodiments, the present disclosure teachesmethods for targeting mutations to a specific locus. In otherembodiments, the present disclosure teaches the use of gene editingtechnologies such as ZFNs, TALENS, or CRISPR, to selectively edit targetDNA regions.

In other embodiments, the present disclosure teaches mutating selectedDNA regions outside of the host organism, and then inserting the mutatedsequence back into the host organism. For example, in some embodiments,the present disclosure teaches mutating native or synthetic promoters toproduce a range of promoter variants with various expression properties(see promoter ladder infra). In other embodiments, the presentdisclosure is compatible with single gene optimization techniques, suchas ProSAR (Fox et al. 2007. “Improving catalytic function byProSAR-driven enzyme evolution.” Nature Biotechnology Vol 25 (3)338-343, incorporated by reference herein).

In some embodiments, the selected regions of DNA are produced in vitrovia gene shuffling of natural variants, or shuffling with syntheticoligos, plasmid-plasmid recombination, virus plasmid recombination,virus-virus recombination. In other embodiments, the genomic regions areproduced via error-prone PCR.

In some embodiments, generating mutations in selected genetic regions isaccomplished by “reassembly PCR.” Briefly, oligonucleotide primers(oligos) are synthesized for PCR amplification of segments of a nucleicacid sequence of interest, such that the sequences of theoligonucleotides overlap the junctions of two segments. The overlapregion is typically about 10 to 100 nucleotides in length. Each of thesegments is amplified with a set of such primers. The PCR products arethen “reassembled” according to assembly protocols. In brief, in anassembly protocol, the PCR products are first purified away from theprimers, by, for example, gel electrophoresis or size exclusionchromatography. Purified products are mixed together and subjected toabout 1-10 cycles of denaturing, reannealing, and extension in thepresence of polymerase and deoxynucleoside triphosphates (dNTP's) andappropriate buffer salts in the absence of additional primers(“self-priming”). Subsequent PCR with primers flanking the gene are usedto amplify the yield of the fully reassembled and shuffled genes.

In some embodiments of the disclosure, mutated DNA regions, such asthose discussed above, are enriched for mutant sequences so that themultiple mutant spectrum, i.e. possible combinations of mutations, ismore efficiently sampled. In some embodiments, mutated sequences areidentified via a mutS protein affinity matrix (Wagner et al., NucleicAcids Res. 23(19):3944-3948 (1995); Su et al., Proc. Natl. Acad. Sci.(U.S.A.), 83:5057-5061(1986)) with a preferred step of amplifying theaffinity-purified material in vitro prior to an assembly reaction. Thisamplified material is then put into an assembly or reassembly PCRreaction as described in later portions of this application.

Promoter Ladders

Promoters regulate the rate at which genes are transcribed and caninfluence transcription in a variety of ways. Constitutive promoters,for example, direct the transcription of their associated genes at aconstant rate regardless of the internal or external cellularconditions, while regulatable, tunable or inducible promoters increaseor decrease the rate at which a gene is transcribed depending on theinternal and/or the external cellular conditions, e.g. growth rate,temperature, responses to specific environmental chemicals, and thelike. Promoters can be isolated from their normal cellular contexts andengineered to regulate the expression of virtually any gene, enablingthe effective modification of cellular growth, product yield and/orother phenotypes of interest.

Promoter sequences can be operably linked to the 5′ termini of anysequences provided herein to be expressed in a filamentous fungal hostcell as provided herein. A variety of known fungal promoters are likelyto be functional in the host strains of the disclosure such as, forexample, the promoter sequences of C1 endoglucanases, the 55 kDacellobiohydrolase (CBH1), glyceraldehyde-3-phosphate dehydrogenase A, C.lucknowense GARG 27K and the 30 kDa xylanase (Xy1F) promoters fromChrysosporium, as well as the Aspergillus promoters described in, e.g.U.S. Pat. Nos. 4,935,349; 5,198,345; 5,252,726; 5,705,358; and5,965,384; and PCT application WO 93/07277. In one embodiment, thepromoters for use in the methods and systems provided herein areinducible promoters. The inducible promoters can be any promoter whosetranscriptional activity is regulated by the presence or absence of achemical such as for example, alcohol, tetracycline, steroids, metal orother compounds known in the art. The inducible promoters can be anypromoter whose transcriptional activity is regulated by the presence orabsence of light or low or high temperatures. In one embodiment, theinducible promoters are selected from filamentous fungal genes such asthe srpB gene, the amyB gene, the manB gene or the mbfA gene. In oneembodiment, the inducible promoter is selected form the promoters listedin Table 1. In one embodiment, the inducible promoter is cataboliterepressed by glucose. The catabolite repressed by glucose can be theamyB promoter from A. oryzae (see FIG. 37).

In some embodiments, the present disclosure teaches methods forproducing promoter ladder libraries for use in downstream genetic designmethods. For example, in some embodiments, the present disclosureteaches methods of identifying one or more promoters and/or generatingvariants of one or more promoters within a host cell, which exhibit arange of expression strengths, or superior regulatory properties. Aparticular combination of these identified and/or generated promoterscan be grouped together as a promoter ladder, which is explained in moredetail below.

In some embodiments, the present disclosure teaches the use of promoterladders. In some embodiments, the promoter ladders of the presentdisclosure comprise promoters exhibiting a continuous range ofexpression profiles. For example, in some embodiments, promoter laddersare created by: identifying natural, native, or wild-type promoters thatexhibit a range of expression strengths in response to a stimuli, orthrough constitutive expression (see e.g., FIG. 13). These identifiedpromoters can be grouped together as a promoter ladder.

In other embodiments, the present disclosure teaches the creation ofpromoter ladders exhibiting a range of expression profiles acrossdifferent conditions. For example, in some embodiments, the presentdisclosure teaches creating a ladder of promoters with expression peaksspread throughout the different stages of a fermentation. In otherembodiments, the present disclosure teaches creating a ladder ofpromoters with different expression peak dynamics in response to aspecific stimulus (see e.g., FIG. 13). Persons skilled in the art willrecognize that the regulatory promoter ladders of the present disclosurecan be representative of any one or more regulatory profiles.

In some embodiments, the promoter ladders of the present disclosure aredesigned to perturb gene expression in a predictable manner across acontinuous range of responses. In some embodiments, the continuousnature of a promoter ladder confers strain improvement programs withadditional predictive power. For example, in some embodiments, swappingpromoters or termination sequences of a selected metabolic or signalingpathway can produce a host cell performance curve, which identifies themost optimum expression ratio or profile; producing a strain in whichthe targeted gene is no longer a limiting factor for a particularreaction or genetic cascade, while also avoiding unnecessary overexpression or misexpression under inappropriate circumstances. Anexample signaling pathway that can be selected can be a signalingpathway that has been identified to or is suspected of playing a role incontrolling or affecting host cell morphology. Accordingly, in someembodiments, swapping promoters for a gene shown to or suspected ofcontrolling or affecting morphology can produce a host cell performancecurve with respect to morphology, which identifies the most optimumexpression ratio or profile of a specific gene for producing a strain orhost cell with a desired pellet morphology under the desired growthcondition; producing a strain in which the targeted gene is no longer alimiting factor for a particular reaction or genetic cascade, while alsoavoiding unnecessary over expression or misexpression underinappropriate circumstances. Examples of genes shown to or suspected ofcontrolling or affecting morphology can be any such genes known in theart or provided herein. In some embodiments, promoter ladders arecreated by: identifying natural, native, or wild-type promotersexhibiting the desired profiles. In other embodiments, the promoterladders are created by mutating naturally occurring promoters to derivemultiple mutated promoter sequences. Each of these mutated promoters istested for effect on target gene expression. In some embodiments, theedited promoters are tested for expression activity across a variety ofconditions, such that each promoter variant's activity isdocumented/characterized/annotated and stored in a database. Theresulting edited promoter variants are subsequently organized intopromoter ladders arranged based on the strength of their expression(e.g., with highly expressing variants near the top, and attenuatedexpression near the bottom, therefore leading to the term “ladder”).

In some embodiments, the present disclosure teaches promoter laddersthat are a combination of identified naturally occurring promoters andmutated variant promoters.

In some embodiments, the present disclosure teaches methods ofidentifying natural, native, or wild-type promoters that satisfied bothof the following criteria: 1) represented a ladder of constitutivepromoters; and 2) could be encoded by short DNA sequences, ideally lessthan 100 base pairs. In some embodiments, constitutive promoters of thepresent disclosure exhibit constant gene expression across two selectedgrowth conditions (typically compared among conditions experiencedduring industrial cultivation). In some embodiments, the promoters ofthe present disclosure will consist of a ˜60 base pair core promoter,and a 5′ UTR between 26- and 40 base pairs in length.

In some embodiments, one or more of the aforementioned identifiednaturally occurring promoter sequences are chosen for gene editing. Insome embodiments, the natural promoters are edited via any of themutation methods described supra. In other embodiments, the promoters ofthe present disclosure are edited by synthesizing new promoter variantswith the desired sequence.

The entire disclosures of U.S. Patent Application No. 62/264,232, filedon Dec. 7, 2015, and International Application No. PCT/US2016/06564,filed on Dec. 7, 2016, are hereby incorporated by reference in itsentirety for all purposes

A non-exhaustive list of the promoters of the present disclosure isprovided in the below Table 1. Each of the promoter sequences can bereferred to as a heterologous promoter or heterologous promoterpolynucleotide.

TABLE 1 Selected promoter sequences of the present disclosure. SEQ IDNO: Promoter Short Name Promoter Name 1 manBp manB promoter fromAspergillus niger 2 amyBp amyB gene from Aspergillus oryzae 3 srpBp srpBpromoter from Aspergillus niger 4 mbfAp mbfA promoter from Aspergillusniger

In some embodiments, the promoters of the present disclosure exhibit atleast 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%,87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, or 75%sequence identity with a promoter from the above table 1.

Terminator Ladders

In some embodiments, the present disclosure teaches methods of improvinggenetically engineered host strains by providing one or moretranscriptional termination sequences at a position 3′ to the end of theRNA encoding element. In some embodiments, the present disclosureteaches that the addition of termination sequences improves theefficiency of RNA transcription of a selected gene in the geneticallyengineered host. In other embodiments, the present disclosure teachesthat the addition of termination sequences reduces the efficiency of RNAtranscription of a selected gene in the genetically engineered host.Thus in some embodiments, the terminator ladders of the presentdisclosure comprises a series of terminator sequences exhibiting a rangeof transcription efficiencies (e.g., one weak terminator, one averageterminator, and one strong promoter).

A transcriptional termination sequence may be any nucleotide sequence,which when placed transcriptionally downstream of a nucleotide sequenceencoding an open reading frame, causes the end of transcription of theopen reading frame. Such sequences are known in the art and may be ofprokaryotic, eukaryotic or phage origin. Examples of terminatorsequences include, but are not limited to, PTH-terminator, pET-T7terminator, T3-Tφ terminator, pBR322-P4 terminator, vesicular stomatitusvirus terminator, rrnB-T1 terminator, rrnC terminator, TTadctranscriptional terminator, and yeast-recognized termination sequences,such as Matα (α-factor) transcription terminator, native α-factortranscription termination sequence, ADR1transcription terminationsequence, ADH2transcription termination sequence, and GAPD transcriptiontermination sequence. A non-exhaustive listing of transcriptionalterminator sequences may be found in the iGEM registry, which isavailable at: partsregistry.org/Terminators/Catalog.

In some embodiments, transcriptional termination sequences may bepolymerase-specific or nonspecific, however, transcriptional terminatorsselected for use in the present embodiments should form a ‘functionalcombination’ with the selected promoter, meaning that the terminatorsequence should be capable of terminating transcription by the type ofRNA polymerase initiating at the promoter. For example, in someembodiments, the present disclosure teaches a eukaryotic RNA pol IIpromoter and eukaryotic RNA pol II terminators, a T7 promoter and T7terminators, a T3 promoter and T3 terminators, a yeast-recognizedpromoter and yeast-recognized termination sequences, etc., wouldgenerally form a functional combination. The identity of thetranscriptional termination sequences used may also be selected based onthe efficiency with which transcription is terminated from a givenpromoter. For example, a heterologous transcriptional terminatorsequence may be provided transcriptionally downstream of the RNAencoding element to achieve a termination efficiency of at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% from a givenpromoter.

In some embodiments, efficiency of RNA transcription from the engineeredexpression construct can be improved by providing nucleic acid sequenceforms a secondary structure comprising two or more hairpins at aposition 3′ to the end of the RNA encoding element. Not wishing to bebound by a particular theory, the secondary structure destabilizes thetranscription elongation complex and leads to the polymerase becomingdissociated from the DNA template, thereby minimizing unproductivetranscription of non-functional sequence and increasing transcription ofthe desired RNA. Accordingly, a termination sequence may be providedthat forms a secondary structure comprising two or more adjacenthairpins. Generally, a hairpin can be formed by a palindromic nucleotidesequence that can fold back on itself to form a paired stem region whosearms are connected by a single stranded loop. In some embodiments, thetermination sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreadjacent hairpins. In some embodiments, the adjacent hairpins areseparated by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15unpaired nucleotides. In some embodiments, a hairpin stem comprises 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or more base pairs in length. In certainembodiments, a hairpin stem is 12 to 30 base pairs in length. In certainembodiments, the termination sequence comprises two or more medium-sizedhairpins having stem region comprising about 9 to 25 base pairs. In someembodiments, the hairpin comprises a loop-forming region of 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the loop-formingregion comprises 4-8 nucleotides. Not wishing to be bound by aparticular theory, stability of the secondary structure can becorrelated with termination efficiency. Hairpin stability is determinedby its length, the number of mismatches or bulges it contains and thebase composition of the paired region. Pairings between guanine andcytosine have three hydrogen bonds and are more stable compared toadenine-thymine pairings, which have only two. The G/C content of ahairpin-forming palindromic nucleotide sequence can be at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90% or more. In some embodiments, the G/C content of ahairpin-forming palindromic nucleotide sequence is at least 80%. In someembodiments, the termination sequence is derived from one or moretranscriptional terminator sequences of prokaryotic, eukaryotic or phageorigin. In some embodiments, a nucleotide sequence encoding a series of4, 5, 6, 7, 8, 9, 10 or more adenines (A) are provided 3′ to thetermination sequence.

In some embodiments, the present disclosure teaches the use of a seriesof tandem termination sequences. In some embodiments, the firsttranscriptional terminator sequence of a series of 2, 3, 4, 5, 6, 7, ormore may be placed directly 3′ to the final nucleotide of the dsRNAencoding element or at a distance of at least 1-5, 5-10, 10-15, 15-20,20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-100, 100-150, 150-200,200-300, 300-400, 400-500, 500-1,000 or more nucleotides 3′ to the finalnucleotide of the dsRNA encoding element. The number of nucleotidesbetween tandem transcriptional terminator sequences may be varied, forexample, transcriptional terminator sequences may be separated by 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40,40-45, 45-50 or more nucleotides. In some embodiments, thetranscriptional terminator sequences may be selected based on theirpredicted secondary structure as determined by a structure predictionalgorithm. Structural prediction programs are well known in the art andinclude, for example, CLC Main Workbench.

Persons having skill in the art will recognize that the methods of thepresent disclosure are compatible with any termination sequence. Anon-exhaustive listing of transcriptional terminator sequences of thepresent disclosure is provided in Table 1.1 below. In one embodiment, atranscriptional terminator of the present disclosure can be anorthologue of a termination sequence provided in Table 1.1. For example,if the host cell is an Aspergillus, the termination sequence can be anorthologue of a non-Aspergillus termination sequence selected from Table1.1.

TABLE 1.1 Non-exhaustive list of termination sequences of the presentdisclosure. Yeast and other Eukaryotes Name Description Direction LengthBBa_J63002 ADH1 terminator from S. cerevisiae Forward 225 BBa_K110012STE2 terminator Forward 123 BBa_K1462070 cyc1 250 BBa_K1486025 ADH1Terminator Forward 188 BBa_K392003 yeast ADH1 terminator 129 BBa_K801011TEF1 yeast terminator 507 BBa_K801012 ADH1 yeast terminator 349BBa_Y1015 CycE1 252 BBa_J52016 eukaryotic -- derived from SV40 earlyForward 238 poly A signal sequence BBa_J63002 ADH1 terminator from S.cerevisiae Forward 225 BBa_K110012 STE2 terminator Forward 123BBa_K1159307 35S Terminator of Cauliflower Mosaic 217 Virus (CaMV)BBa_K1462070 cyc1 250 BBa_K1484215 nopaline synthase terminator 293BBa_K1486025 ADH1 Terminator Forward 188 BBa_K392003 yeast ADH1terminator 129 BBa_K404108 hGH terminator 481 BBa_K404116hGH_[AAV2]-right-ITR 632 BBa_K678012 SV40 poly A, terminator for 139mammalian cells BBa_K678018 hGH poly A, terminator for mammalian 635cells BBa_K678019 BGH poly A, mammalian terminator 233 BBa_K678036 trpCterminator for Aspergillus 759 nidulans BBa_K678037 T1-motni, terminatorfor Aspergillus 1006 niger BBa_K678038 T2-motni, terminator forAspergillus 990 niger BBa_K678039 T3-motni, terminator for Aspergillus889 niger BBa_K801011 TEF1 yeast terminator 507 BBa_K801012 ADH1 yeastterminator 349 BBa_Y1015 CycE1 252Hypothesis-Driven Diversity Pools and Hill Climbing

The present disclosure teaches that the HTP genomic engineering methodsof the present disclosure do not require prior genetic knowledge inorder to achieve significant gains in host cell performance. Indeed, thepresent disclosure teaches methods of generating diversity pools viaseveral functionally agnostic approaches, including random mutagenesis,and identification of genetic diversity among pre-existing host cellvariants (e.g., such as the comparison between a wild type host cell andan industrial variant).

In some embodiments however, the present disclosure also teacheshypothesis-driven methods of designing genetic diversity mutations thatwill be used for downstream HTP engineering. That is, in someembodiments, the present disclosure teaches the directed design ofselected mutations. In some embodiments, the directed mutations areincorporated into the engineering libraries of the present disclosure(e.g., SNP swap, PRO swap, or STOP swap).

In some embodiments, the present disclosure teaches the creation ofdirected mutations based on gene annotation, hypothesized (or confirmed)gene function, or location within a genome. The diversity pools of thepresent disclosure may include mutations in genes hypothesized to beinvolved in a specific metabolic or genetic pathway associated in theliterature with increased performance of a host cell. In otherembodiments, the diversity pool of the present disclosure may alsoinclude mutations to genes associated with improved host performance. Inyet other embodiments, the diversity pool of the present disclosure mayalso include mutations to genes based on algorithmic predicted function,or other gene annotation.

In some embodiments, the present disclosure teaches a “shell” basedapproach for prioritizing the targets of hypothesis-driven mutations.The shell metaphor for target prioritization is based on the hypothesisthat only a handful of primary genes are responsible for most of aparticular aspect of a host cell's performance (e.g., production of asingle biomolecule). These primary genes are located at the core of theshell, followed by secondary effect genes in the second layer, tertiaryeffects in the third shell, and . . . etc. For example, in oneembodiment the core of the shell might comprise genes encoding criticalbiosynthetic enzymes within a selected metabolic pathway (e.g.,production of citric acid). Genes located on the second shell mightcomprise genes encoding for other enzymes within the biosyntheticpathway responsible for product diversion or feedback signaling. Thirdtier genes under this illustrative metaphor would likely compriseregulatory genes responsible for modulating expression of thebiosynthetic pathway, or for regulating general carbon flux within thehost cell.

The present disclosure also teaches “hill climb” methods for optimizingperformance gains from every identified mutation. In some embodiments,the present disclosure teaches that random, natural, orhypothesis-driven mutations in HTP diversity libraries can result in theidentification of genes associated with host cell performance. Forexample, the present methods may identify one or more beneficial SNPslocated on, or near, a gene coding sequence. This gene might beassociated with host cell performance, and its identification can beanalogized to the discovery of a performance “hill” in the combinatorialgenetic mutation space of an organism.

In some embodiments, the present disclosure teaches methods of exploringthe combinatorial space around the identified hill embodied in the SNPmutation. That is, in some embodiments, the present disclosure teachesthe perturbation of the identified gene and associated regulatorysequences in order to optimize performance gains obtained from that genenode (i.e., hill climbing). Thus, according to the methods of thepresent disclosure, a gene might first be identified in a diversitylibrary sourced from random mutagenesis, but might be later improved foruse in the strain improvement program through the directed mutation ofanother sequence within the same gene.

The concept of hill climbing can also be expanded beyond the explorationof the combinatorial space surrounding a single gene sequence. In someembodiments, a mutation in a specific gene might reveal the importanceof a particular metabolic or genetic pathway to host cell performance.For example, in some embodiments, the discovery that a mutation in asingle RNA degradation gene resulted in significant host performancegains could be used as a basis for mutating related RNA degradationgenes as a means for extracting additional performance gains from thehost organism. Persons having skill in the art will recognize variantsof the above described shell and hill climb approaches to directedgenetic design.

Morphology-Related Genes

The morphology related genes for use in the methods, strains and systemsprovided herein can be any gene known in the art that has been shown oris suspected to play a role in controlling or affecting the morphologyof a filamentous eukaryotic microbe (e.g., filamentous fungal host cellor strain). The gene that regulates morphology of the host cell can beany such gene as provided herein. In one embodiment, the gene is anorthologue of the S. cerevisiae SLN1. In another embodiment, themorphology related gene can be any gene from the same pathway as theorthologue of the S. cerevisiae SLN1 gene. The genes that are part ofthe same pathway can be selected from orthologues of the S. cerevisiaeYpd1, Skn7, Ssk1 and Ssk2 genes or any combination thereof. In anotherembodiment, the gene is an orthologue of the A. niger gene with nucleicacid SEQ ID NO: 11 and/or any gene in the same biochemical pathway ofsaid orthologue of the A. niger gene with nucleic acid SEQ ID NO: 11. Inanother embodiment, the gene is an orthologue of the A. niger gene withnucleic acid SEQ ID NO: 12 and/or any gene in the same biochemicalpathway of said orthologue of the A. niger gene with nucleic acid SEQ IDNO: 12. In another embodiment, the gene is an orthologue of the A. nigergene with nucleic acid SEQ ID NO. 14 and/or any gene in the samebiochemical pathway of said orthologue of the A. niger gene with nucleicacid SEQ ID NO: 14.

The morphology related genes for use in the methods, strains and systemsprovided herein can be any gene known in the art that has been shown oris suspected to play a role in controlling or affecting the morphologyof A. niger. In one embodiment, the gene is a SNP containing gene with anucleic acid sequence selected from SEQ ID NOs: 11, 12, 13 or 14 (seeTable 4). In one embodiment, the gene is a plurality of genes. Theplurality of genes can be any combination of the SNP containing geneswith a nucleic acid sequence selected from SEQ ID NOs: 11, 12, 13 or 14.The plurality of genes can be any combination of the SNP containinggenes with a nucleic acid sequence selected from SEQ ID NOs: 11 and anygene present within the same biochemical pathway. The plurality of genescan be any combination of the SNP containing genes with a nucleic acidsequence selected from SEQ ID NOs: 12 and any gene present within thesame biochemical pathway. The plurality of genes can be any combinationof the SNP containing genes with a nucleic acid sequence selected fromSEQ ID NOs: 13 and any gene present within the same biochemical pathway.The plurality of genes can be any combination of the SNP containinggenes with a nucleic acid sequence selected from SEQ ID NOs: 14 and anygene present within the same biochemical pathway. In one embodiment, thegene is a wild-type or non-SNP containing version of the gene with anucleic acid sequence selected from SEQ ID NOs: 11, 12, 13 or 14 (seeTable 4).

In one embodiment, the gene that regulates morphology of an A. nigerhost cell is an A. niger orthologue of the S. cerevisiae SLN1 gene. TheA. niger ortholog of the S. cerevisiae SLN1 gene can be a wild-type formor a mutant form. The mutated form of the A. niger orthologue of the S.cerevisiae SLN1 gene can be FungiSNP_18 from Table 4 or with a nucleicacid sequence of SEQ ID NO: 13. In another embodiment, the morphologyrelated gene can be any gene from the same pathway as the A. nigerorthologue of the S. cerevisiae SLN1 gene. The genes that are part ofthe same pathway can be selected from A. niger orthologues of the S.cerevisiae Ypd1, Skn7, Ssk1 and Ssk2 genes or any combination thereof.The genes that are part of the same pathway can be selected from thenucleic acid sequences represented by SEQ ID NOs: 15, 16, 17, 18, 19 orany combination thereof.

The morphology-related genes can be any of the genes or orthologuesthereof that are disclosed in Dai et al. (“Identification of GenesAssociated with Morphology in Aspergillus niger by Using SuppressionSubtractive Hybridization” Applied and Environmental Microbiology, April2004, p. 2474-2485), the contents of which are incorporated by referencein its entirety. The morphology-related gene can be selected from thegas1 gene, the sfb3 gene, the seb1 gene, the mpg1 gene, the crz1 gene,and the tps2 gene. The expression of any of the morphology related genescan be increased or decreased depending on if the gene promotes afilamentous or mycelial morphology or pellet morphology.

As described herein, the expression of any of the morphology relatedgenes or mutant thereof (e.g., FungiSNPs 9, 12, 18 or 40 from Table 4)provided herein can be controlled by replacing the native promoter ofthe gene with a heterologous promoter that confers expression at a level(e.g., higher or lower) different from the native promoter. Theheterologous promoter can be selected from Table 1. Replacement of thenative promoter can be performed using a PRO swap method as providedherein.

It is a further object of the present invention to provide a filamentousfungus host cell comprising a heterologous modification of the hostcell's orthologue of a S. cerevisiae SLN1 gene, whereby the modifiedorthologue of the S. cerevisiae SLN1 gene has reduced activity and/orreduced expression relative to a parental filamentous fungal host celllacking the heterologous modification. The filamentous fungal host canpossess a non-mycelium, pellet forming phenotype. This pellet phenotypecan be due to the filamentous fungal host cell possessing theheterologous modification in the orthologue of the S. cerevisiae SLN1gene that causes cells of the filamentous host cell to produce asubstantially reduced amount and/or substantially less active form offunctional orthologue of a S. cerevisiae SLN1 gene as compared to cellsof that do not possess said heterologous modification. The filamentousfungal host cell and any parental strain said filamentous fungal hostcell is derived therefrom can be any filamentous fungus known in the artand/or provided herein such as, for example, A. niger. In oneembodiment, the filamentous fungal host cell sporulates normally ascompared to a parental strain when grown under non-submerged growthconditions such as, for example, on solid media. In another embodiment,the filamentous fungal host cell is sporulates normally as compared tothe parental strain when grown under non-submerged growth conditionssuch as, for example, on solid media only when one, all or a combinationof orthologoues of the SNP containing gene from Table 4 are alsoexpressed in the filamentous fungal host cell. In one embodiment, thefilamentous fungal host cell is A. niger and said A. niger host cellsporulates normally as compared to a parental strain when grown undernon-submerged growth conditions such as, for example, on solid mediaonly when one, all or a combination of the SNP containing genes fromTable 4 are also expressed in said A. niger host cell. In yet anotherembodiment, the filamentous fungal host cell sporulates normally ascompared to a parental strain when grown under non-submerged growthconditions such as, for example, on solid media only when one, all or acombination of orthologoues of the SNP containing genes from Table 4 arealso expressed in the filamentous fungal host cell. The submergedculture conditions can comprise growing the variant strain in CAPmedium. The CAP media can comprise manganese and be substantially freeof chelating agents. The manganese can be present in amount that is atleast 13 ppb or higher.

The genetic alteration to the orthologue of the S. cerevisiae SLN1 genecan be replacement of the wild-type form of the gene with a mutatedorthologue of the S. cerevisiae SLN1 gene, replacement of the nativepromoter of the gene with a heterologous promoter that more weaklyexpresses the gene for the orthologue of the S. cerevisiae SLN1 proteinas compared to the native promoter, or a combination thereof.Alternatively, the genetic alteration to the orthologue of the S.cerevisiae SLN1 gene can be the removal of the orthologue of the S.cerevisiae SLN1 gene and replacement with a selectable marker gene. Themutated form of the orthologue of the S. cerevisiae SLN1 gene cancomprise a SNP, a non-sense mutation, a missense mutation, a deletion,an insertion or any combination thereof. In one embodiment, thefilamentous fungal host cell is A. niger and the A. niger orthologue ofthe S. cerevisiae SLN1 protein can be encoded by SEQ ID NO: 13. Theheterologous promoter can be selected from a promoter listed in Table 1.In one embodiment, the heterologous promoter is a manB or amyB promoter.Further to this embodiment, the heterologous promoter can be SEQ ID NO:1 or SEQ ID NO: 2. The selectable marker can be selected from anauxotrophic marker gene, a colorimetric marker gene, antibioticresistance gene, or a directional marker gene as provided herein.

The filamentous fungal host cell that possesses a substantially reducedamount and/or substantially less active form of functional orthologue ofthe S. cerevisiae SLN1 protein can further comprise a genetic disruptionor alteration in one or more genes that are part of the same pathway asthe orthologue of the S. cerevisiae SLN1 gene. The one or more genesthat are part of the same pathway can be selected from orthologues ofthe S. cerevisiae Ypd1, Skn7, Ssk1 and Ssk2 genes or any combinationthereof. In one embodiment, the filamentous fungal host cell is A. nigerand the orthologues of the S. cerevisiae SLN1, Ypd1, Skn7, Ssk1 and Ssk2genes are A. niger orthologues or mutants thereof. Further to thisembodiment, the one or more genes that are part of the same pathway canbe selected from the nucleic acid sequences represented by SEQ ID NOs:15, 16, 17, 18, 19 or any combination thereof. The filamentous fungalhost cell can further comprise a genetic disruption or alteration in oneor more genes that are part of the different pathway that is known toplay a role in controlling filamentous fungal morphology. The one ormore genes that are part of the different pathway can be any of thegenes provided herein. The one or more genes that are part of thedifferent pathway can be selected from A. niger orthologues of geneswith nucleic acid sequences represented by SEQ ID NOs: 11, 12, 14 or anycombination thereof. In one embodiment, the filamentous fungal host cellis A. niger and the one or more genes that are part of the differentpathway are the A. niger genes with nucleic acid sequences representedby SEQ ID NOs: 11, 12, 14 or any combination thereof. In anotherembodiment, the filamentous fungal host cell is A. niger and the one ormore genes that are part of the different pathway are the non-SNPcontaining versions of the A. niger genes with nucleic acid sequencesrepresented by SEQ ID NOs: 11, 12, 14 or any combination thereof.

The genetic disruption or alteration to the one or more genes that arepart of the same pathway as the orthologue of the S. cerevisiae SLN1gene or are part of the different pathway that is known to play a rolein controlling filamentous fungal morphology can be replacement of thewild-type form of the gene with a mutated form of the gene, replacementof the native promoter of the gene with a heterologous promoter thatalters the expression (e.g., higher or lower) of the gene as compared tothe native promoter, or a combination thereof. The promoter can be apromoter listed in Table 1. Alternatively, the genetic disruption oralteration to the one or more genes that are part of the same pathway asthe orthologue of the S. cerevisiae SLN1 gene or are part of thedifferent pathway that is known to play a role in controllingfilamentous fungal morphology can be the removal of the gene andreplacement with a selectable marker gene. The selectable marker can beselected from an auxotrophic marker gene, a colorimetric marker gene,antibiotic resistance gene, or a directional marker gene as providedherein.

Also provided herein, are methods for generating the filamentous fungushost cell that possess a substantially reduced amount and/orsubstantially less active form of functional orthologue of the S.cerevisiae SLN1 protein. The methods can comprise performing a PRO swapmethod, a SNP Swap method or a combination of a PRO swap and SNP swapmethod as provided herein.

It is a further object of the present invention to provide a filamentousfungus host cell comprising a heterologous modification of the hostcell's orthologue of an A. niger gene with a nucleic acid sequenceselected from SEQ ID NO. 11, 12, 14 or any combination thereof, wherebythe modified orthologue of the A. niger gene with a nucleic acidsequence selected from SEQ ID NO. 11, 12, 14 or any combination thereofhas reduced activity and/or reduced expression relative to a parentalfilamentous fungal host cell lacking the heterologous modification(s).The filamentous fungal host can possess a non-mycelium, pellet formingphenotype as compared to the cells of the parental strain when grown ina submerged culture due to the filamentous host cell possessing aheterologous modification to the orthologue of an A. niger gene withnucleic acid sequence SEQ ID NO: 11, 12, 14 or any combination thereof.Possession of an orthologue of an A. niger gene with a nucleic acidsequence of SEQ ID NO: 11, 12, 14 or any combination thereof can causecells of the host cell to produce a substantially reduced amount and/orsubstantially less active form of functional protein encoded byorthologues of the A. niger genes with said SEQ ID NOs as compared tocells of a parental host cell when grown under submerged cultureconditions. The filamentous host cell and parental strain of saidfilamentous fungal host cell can be any filamentous fungus known in theart and/or provided herein such as, for example, A. niger. In oneembodiment, the filamentous host cell strain sporulates normally ascompared to a parental strain when grown under non-submerged growthconditions such as, for example, on solid media. In some cases, theorthologues of the A. niger genes with SEQ ID NOs; 11, 12 or 14 arefurther genetically altered. The further genetic alteration can bereplacement of the native promoter of the gene with a heterologouspromoter that more weakly expresses the gene as compared to the nativepromoter. Alternatively, the further genetic alteration can be theremoval of the orthologues of the A. niger genes with SEQ ID NO: 11, 12or 14 and replacement with a selectable marker gene. The selectablemarker can be selected from an auxotrophic marker gene, a colorimetricmarker gene, antibiotic resistance gene, or a directional marker gene asprovided herein. The heterologous promoter can be selected from apromoter listed in Table 1. In one embodiment, the heterologous promoteris a manB or amyB promoter. Further to this embodiment, the heterologouspromoter can have the nucleic acid sequence of SEQ ID NO: 1 or SEQ IDNO: 2. The submerged culture conditions can comprise growing the variantstrain in CAP medium. The CAP media can comprise manganese and besubstantially free of chelating agents. The manganese can be present inamount that is at least 13 ppb or higher. It should be understood thatin embodiments where the filamentous fungal host cell is A. niger, theA. niger gene with a nucleic acid sequence selected from SEQ ID NO. 11,12, 14 or wild-type versions thereof can comprise the heterologousmodifications detailed herein.

The filamentous fungal host cell that possesses a substantially reducedamount and/or substantially less active form of functional proteinencoded by orthologues of the A. niger genes with sequences selectedfrom SEQ ID NOs: 11, 12 or 14 can further comprise a genetic disruptionor alteration in one or more genes that are part of the same pathway.The filamentous fungal host cell can further comprise a geneticdisruption or alteration in one or more genes that are part of thedifferent pathway that is known to play a role in controllingfilamentous fungal morphology. The one or more genes that are part ofthe different pathway can be any of the genes provided herein. Thegenetic disruption or alteration to the one or more genes that are partof the same pathway or are part of the different pathway that is knownto play a role in controlling filamentous fungal morphology can bereplacement of the wild-type form of the gene with a mutated form of thegene, replacement of the native promoter of the gene with a heterologouspromoter that alters the expression (e.g., higher or lower) of the geneas compared to the native promoter, or a combination thereof. Thepromoter can be a promoter listed in Table 1. Alternatively, the geneticdisruption or alteration to the one or more genes that are part of thesame pathway or are part of the different pathway that is known to playa role in controlling filamentous fungal morphology can be the removalof the gene and replacement with a selectable marker gene. Theselectable marker can be selected from an auxotrophic marker gene, acolorimetric marker gene, antibiotic resistance gene, or a directionalmarker gene as provided herein.

Also provided herein, are methods for generating the variant strain offilamentous fungus that possess a substantially reduced amount and/orsubstantially less active form of functional protein encoded byorthologues of the A. niger genes with SEQ ID NOs: 11, 12 or 14. Themethods can comprise performing a PRO swap method, a SNP Swap method ora combination of a PRO swap and SNP swap method as provided herein.

It is yet another object of this invention to provide a filamentousfungal host cell comprising a promoter operably linked to a gene thatregulates morphology of the host cell, wherein the promoter isheterologous to the gene, and wherein the promoter has a sequenceselected from the group consisting of SEQ ID NOs. 1-4. The filamentousfungus host cell can be any filamentous fungus known in the art and/orprovided herein such as, for example, A. niger. In some cases, thefungal host cell sporulates normally as compared to a parental strain ofthe host cell when grown under non-submerged growth conditions such as,for example, on solid media, but forms a non-mycelium, pellet morphologywhen grown under submerged culture conditions. In some cases, the hostcell can comprise one or more genes that regulate morphology such thateach of said one or more genes has a heterologous promoter linkedthereto. The one or more genes that regulates morphology of the hostcell can be any such gene as provided herein such as, for example, theSNP containing gene sequences represented by SEQ ID NOs: 11, 12, 13 or14 or orthologues thereof from Table 4, either alone or in combination.In some cases, the SNP containing gene sequences represented by SEQ IDNOs: 11, 12, 13 or 14 or orthologues thereof from Table 4 can be incombination with one or more genes from the same pathway as therespective SNP containing gene sequence. In one embodiment, the one ormore genes is a wild-type or non-SNP containing version of the gene witha nucleic acid sequence selected from SEQ ID NOs: 11, 12, 13 or 14 ororthologues thereof, either alone or in combination. In anotherembodiment, the wild-type or non-SNP containing version of the gene witha nucleic acid sequence selected from SEQ ID NOs: 11, 12, 13 or 14 ororthologues thereof can be in combination with one or more genes fromthe same pathway as the respective wild-type or non-SNP containing genesequence. In one embodiment, the gene that regulates morphology of thehost cell can be an orthologue of the S. cerevisiae SLN1 gene or a genein the same signaling pathway. The one or more genes that are part ofthe same signaling pathway can be selected from orthologues of the S.cerevisiae Ypd1, Skn7, Ssk1 and Ssk2 genes or any combination thereof.In one embodiment, the filamentous fungal host cell is A. niger and theone or more genes that are part of the same signaling pathway can beselected from the nucleic acid sequences represented by SEQ ID NOs: 15,16, 17, 18, 19 or any combination thereof. The orthologue of the S.cerevisiae SLN1 gene can be a wild-type or mutant form of the gene. Inone embodiment, the filamentous fungal host cell is A. niger and themutated A. niger orthologue of the S. cerevisiae SLN1 gene has thenucleic acid sequence of SEQ ID NO: 13. The submerged culture conditionscan comprise growing the variant strain in CAP medium. The CAP media cancomprise manganese and be substantially free of chelating agents. Themanganese can be present in amount that is at least 13 ppb or higher.

Cell Culture and Fermentation

Cells of the present disclosure can be cultured in conventional nutrientmedia modified as appropriate for any desired biosynthetic reactions orselections. In some embodiments, the present disclosure teaches culturein inducing media for activating promoters. In some embodiments, thepresent disclosure teaches media with selection agents, includingselection agents of transformants (e.g., antibiotics), or selection oforganisms suited to grow under inhibiting conditions (e.g., high ethanolconditions). In some embodiments, the present disclosure teaches growingcell cultures in media optimized for cell growth. In other embodiments,the present disclosure teaches growing cell cultures in media optimizedfor product yield. In some embodiments, the present disclosure teachesgrowing cultures in media capable of inducing cell growth and alsocontains the necessary precursors for final product production (e.g.,high levels of sugars for ethanol production).

Culture conditions, such as temperature, pH and the like, are thosesuitable for use with the host cell selected for expression, and will beapparent to those skilled in the art. As noted, many references areavailable for the culture and production of many cells, including cellsof bacterial, plant, animal (including mammalian) and archaebacterialorigin. See e.g., Sambrook, Ausubel (all supra), as well as Berger,Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif.; and Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Doyle and Griffiths (1997)Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY;Humason (1979) Animal Tissue Techniques, fourth edition W.H. Freeman andCompany; and Ricciardelle et al., (1989) In Vitro Cell Dev. Biol.25:1016-1024, all of which are incorporated herein by reference. Forplant cell culture and regeneration, Payne et al. (1992) Plant Cell andTissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg N.Y.); Jones, ed. (1984) Plant Gene Transfer and ExpressionProtocols, Humana Press, Totowa, N.J. and Plant Molecular Biology (1993)R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN 0 12198370 6, all of which are incorporated herein by reference. Cellculture media in general are set forth in Atlas and Parks (eds.) TheHandbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.,which is incorporated herein by reference. Additional information forcell culture is found in available commercial literature such as theLife Science Research Cell Culture Catalogue from Sigma-Aldrich, Inc (StLouis, Mo.) (“Sigma-LSRCCC”) and, for example, The Plant CultureCatalogue and supplement also from Sigma-Aldrich, Inc (St Louis, Mo.)(“Sigma-PCCS”), all of which are incorporated herein by reference.

The culture medium to be used must in a suitable manner satisfy thedemands of the respective strains. Descriptions of culture media forvarious microorganisms are present in the “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981).

The present disclosure furthermore provides a process for fermentativepreparation of a product of interest, comprising the steps of: a)culturing a microorganism according to the present disclosure in asuitable medium, resulting in a fermentation broth; and b) concentratingthe product of interest in the fermentation broth of a) and/or in thecells of the microorganism.

In some embodiments, the present disclosure teaches that themicroorganisms produced may be cultured continuously—as described, forexample, in WO 05/021772—or discontinuously in a batch process (batchcultivation) or in a fed-batch or repeated fed-batch process for thepurpose of producing the desired organic-chemical compound. A summary ofa general nature about known cultivation methods is available in thetextbook by Chmiel (Bioprozeßtechnik. 1: Einführung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in thetextbook by Storhas (Bioreaktoren and periphere Einrichtungen (ViewegVerlag, Braunschweig/Wiesbaden, 1994)).

In some embodiments, the cells of the present disclosure are grown underbatch or continuous fermentations conditions.

Classical batch fermentation is a closed system, wherein thecompositions of the medium is set at the beginning of the fermentationand is not subject to artificial alternations during the fermentation. Avariation of the batch system is a fed-batch fermentation which alsofinds use in the present disclosure. In this variation, the substrate isadded in increments as the fermentation progresses. Fed-batch systemsare useful when catabolite repression is likely to inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the medium. Batch and fed-batch fermentationsare common and well known in the art.

Continuous fermentation is a system where a defined fermentation mediumis added continuously to a bioreactor and an equal amount of conditionedmedium is removed simultaneously for processing and harvesting ofdesired biomolecule products of interest. In some embodiments,continuous fermentation generally maintains the cultures at a constanthigh density where cells are primarily in log phase growth. In someembodiments, continuous fermentation generally maintains the cultures ata stationary or late log/stationary, phase growth. Continuousfermentation systems strive to maintain steady state growth conditions.

Methods for modulating nutrients and growth factors for continuousfermentation processes as well as techniques for maximizing the rate ofproduct formation are well known in the art of industrial microbiology.

For example, a non-limiting list of carbon sources for the cultures ofthe present disclosure include, sugars and carbohydrates such as, forexample, glucose, sucrose, lactose, fructose, maltose, molasses,sucrose-containing solutions from sugar beet or sugar cane processing,starch, starch hydrolysate, and cellulose; oils and fats such as, forexample, soybean oil, sunflower oil, groundnut oil and coconut fat;fatty acids such as, for example, palmitic acid, stearic acid, andlinoleic acid; alcohols such as, for example, glycerol, methanol, andethanol; and organic acids such as, for example, acetic acid or lacticacid.

A non-limiting list of the nitrogen sources for the cultures of thepresent disclosure include, organic nitrogen-containing compounds suchas peptones, yeast extract, meat extract, malt extract, corn steepliquor, soybean flour, and urea; or inorganic compounds such as ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, andammonium nitrate. The nitrogen sources can be used individually or as amixture.

A non-limiting list of the possible phosphorus sources for the culturesof the present disclosure include, phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogen phosphate or the correspondingsodium-containing salts.

The culture medium may additionally comprise salts, for example in theform of chlorides or sulfates of metals such as, for example, sodium,potassium, magnesium, calcium and iron, such as, for example, magnesiumsulfate or iron sulfate, which are necessary for growth.

Finally, essential growth factors such as amino acids, for examplehomoserine and vitamins, for example thiamine, biotin or pantothenicacid, may be employed in addition to the abovementioned substances.

In some embodiments, the pH of the culture can be controlled by any acidor base, or buffer salt, including, but not limited to sodium hydroxide,potassium hydroxide, ammonia, or aqueous ammonia; or acidic compoundssuch as phosphoric acid or sulfuric acid in a suitable manner. In someembodiments, the pH is generally adjusted to a value of from 6.0 to 8.5,preferably 6.5 to 8.

In some embodiments, the cultures of the present disclosure may includean anti-foaming agent such as, for example, fatty acid polyglycolesters. In some embodiments the cultures of the present disclosure aremodified to stabilize the plasmids of the cultures by adding suitableselective substances such as, for example, antibiotics.

In some embodiments, the culture is carried out under aerobicconditions. In order to maintain these conditions, oxygen oroxygen-containing gas mixtures such as, for example, air are introducedinto the culture. It is likewise possible to use liquids enriched withhydrogen peroxide. The fermentation is carried out, where appropriate,at elevated pressure, for example at an elevated pressure of from 0.03to 0.2 MPa. The temperature of the culture is normally from 20° C. to45° C. and preferably from 25° C. to 40° C., particularly preferablyfrom 30° C. to 37° C. In batch or fed-batch processes, the cultivationis preferably continued until an amount of the desired product ofinterest (e.g. an organic-chemical compound) sufficient for beingrecovered has formed. This aim can normally be achieved within 10 hoursto 160 hours. In continuous processes, longer cultivation times arepossible. The activity of the microorganisms results in a concentration(accumulation) of the product of interest in the fermentation mediumand/or in the cells of said microorganisms.

In some embodiments, the culture is carried out under anaerobicconditions.

Screening

In some embodiments, the present disclosure teaches high-throughputinitial screenings. In other embodiments, the present disclosure alsoteaches robust tank-based validations of performance data (see FIG. 6B).

In some embodiments, the high-throughput screening process is designedto predict performance of strains in bioreactors. As previouslydescribed, culture conditions are selected to be suitable for theorganism and reflective of bioreactor conditions. Individual coloniesare picked and transferred into 96 well plates and incubated for asuitable amount of time. Cells are subsequently transferred to new 96well plates for additional seed cultures, or to production cultures.Cultures are incubated for varying lengths of time, where multiplemeasurements may be made. These may include measurements of product,biomass or other characteristics that predict performance of strains inbioreactors. High-throughput culture results are used to predictbioreactor performance.

In some embodiments, the tank-based performance validation is used toconfirm performance of strains isolated by high-throughput screening.Candidate strains are screened using bench scale fermentation reactors(e.g., reactors disclosed in Table 3 of the present disclosure) forrelevant strain performance characteristics such as productivity oryield.

Product Recovery and Quantification

Methods for screening for the production of products of interest areknown to those of skill in the art and are discussed throughout thepresent specification. Such methods may be employed when screening thestrains of the disclosure.

In some embodiments, the present disclosure teaches methods of improvingstrains designed to produce non-secreted intracellular products. Forexample, the present disclosure teaches methods of improving therobustness, yield, efficiency, or overall desirability of cell culturesproducing intracellular enzymes, oils, pharmaceuticals, or othervaluable small molecules or peptides. The recovery or isolation ofnon-secreted intracellular products can be achieved by lysis andrecovery techniques that are well known in the art, including thosedescribed herein.

For example, in some embodiments, cells of the present disclosure can beharvested by centrifugation, filtration, settling, or other method.Harvested cells are then disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents, or other methods, which are well known to those skilledin the art.

The resulting product of interest, e.g. a polypeptide, may berecovered/isolated and optionally purified by any of a number of methodsknown in the art. For example, a product polypeptide may be isolatedfrom the nutrient medium by conventional procedures including, but notlimited to: centrifugation, filtration, extraction, spray-drying,evaporation, chromatography (e.g., ion exchange, affinity, hydrophobicinteraction, chromatofocusing, and size exclusion), or precipitation.Finally, high performance liquid chromatography (HPLC) can be employedin the final purification steps. (See for example Purification ofintracellular protein as described in Parry el al., 2001, Biochem. J.353:117, and Hong et al., 2007, Appl. Microbiol. Biotechnol. 73:1331,both incorporated herein by reference).

In addition to the references noted supra, a variety of purificationmethods are well known in the art, including, for example, those setforth in: Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2^(nd) Edition, Wiley-Liss,NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ;Harris and Angal (1990) Protein Purification Applications: A PracticalApproach, IRL Press at Oxford, Oxford, England; Harris and Angal ProteinPurification Methods: A Practical Approach, IRL Press at Oxford, Oxford,England; Scopes (1993) Protein Purification: Principles and Practice3^(rd) Edition, Springer Verlag, NY; Janson and Ryden (1998) ProteinPurification: Principles. High Resolution Methods and Applications,Second Edition, Wiley-VCH, NY; and Walker (1998) Protein Protocols onCD-ROM, Humana Press, NJ, all of which are incorporated herein byreference.

In some embodiments, the present disclosure teaches the methods ofimproving strains designed to produce secreted products. For example,the present disclosure teaches methods of improving the robustness,yield, efficiency, or overall desirability of cell cultures producingvaluable small molecules or peptides.

In some embodiments, immunological methods may be used to detect and/orpurify secreted or non-secreted products produced by the cells of thepresent disclosure. In one example approach, antibody raised against aproduct molecule (e.g., against an insulin polypeptide or an immunogenicfragment thereof) using conventional methods is immobilized on beads,mixed with cell culture media under conditions in which theendoglucanase is bound, and precipitated. In some embodiments, thepresent disclosure teaches the use of enzyme-linked immunosorbent assays(ELISA).

In other related embodiments, immunochromatography is used, as disclosedin U.S. Pat. Nos. 5,591,645, 4,855,240, 4,435,504, 4,980,298, andSe-Hwan Paek, et al., “Development of rapid One-StepImmunochromatographic assay, Methods”, 22, 53-60, 2000), each of whichare incorporated by reference herein. A general immunochromatographydetects a specimen by using two antibodies. A first antibody exists in atest solution or at a portion at an end of a test piece in anapproximately rectangular shape made from a porous membrane, where thetest solution is dropped. This antibody is labeled with latex particlesor gold colloidal particles (this antibody will be called as a labeledantibody hereinafter). When the dropped test solution includes aspecimen to be detected, the labeled antibody recognizes the specimen soas to be bonded with the specimen. A complex of the specimen and labeledantibody flows by capillarity toward an absorber, which is made from afilter paper and attached to an end opposite to the end having includedthe labeled antibody. During the flow, the complex of the specimen andlabeled antibody is recognized and caught by a second antibody (it willbe called as a tapping antibody hereinafter) existing at the middle ofthe porous membrane and, as a result of this, the complex appears at adetection part on the porous membrane as a visible signal and isdetected.

In some embodiments, the screening methods of the present disclosure arebased on photometric detection techniques (absorption, fluorescence).For example, in some embodiments, detection may be based on the presenceof a fluorophore detector such as GFP bound to an antibody. In otherembodiments, the photometric detection may be based on the accumulationon the desired product from the cell culture. In some embodiments, theproduct may be detectable via UV of the culture or extracts from saidculture.

Persons having skill in the art will recognize that the methods of thepresent disclosure are compatible with host cells producing anydesirable biomolecule product of interest. Table 2 below presents anon-limiting list of the product categories, biomolecules, and hostcells, included within the scope of the present disclosure. Theseexamples are provided for illustrative purposes, and are not meant tolimit the applicability of the presently disclosed technology in anyway.

TABLE 2 A non-limiting list of the host cells and products of interestof the present disclosure. Product category Products Host category HostsFlavor & Agarwood Yeast Saccharomyces cerevisiae Fragrance Flavor &Ambrox Yeast Saccharomyces cerevisiae Fragrance Flavor & NootkatoneYeast Saccharomyces cerevisiae Fragrance Flavor & Patchouli oil YeastSaccharomyces cerevisiae Fragrance Flavor & Saffron Yeast Saccharomycescerevisiae Fragrance Flavor & Sandalwood oil Yeast Saccharomycescerevisiae Fragrance Flavor & Valencene Yeast Saccharomyces cerevisiaeFragrance Flavor & Vanillin Yeast Saccharomyces cerevisiae FragranceFood CoQ10/Ubiquinol Yeast Schizosaccharomyces pombe Food Omega 3 fattyMicroalgae Schizochytrium acids Food Omega 6 fatty MicroalgaeSchizochytrium acids Food Vitamin B2 Filamentous Ashbya gossypii fungiFood Erythritol Yeast-like fungi Torula coralline Food ErythritolYeast-like fungi Pseudozyma tsukubaensis Food Erythritol Yeast-likefungi Moniliella pollinis Food Steviol glycosides Yeast Saccharomycescerevisiae Organic acids Citric acid Filamentous Aspergillus niger fungiOrganic acids Citric Acid Filamentous Aspergillus carbonarius fungiOrganic acids Citric Acid Filamentous Aspergillus aculeatus fungiOrganic acids Citric acid Yeast Pichia guilliermondii Organic acidsGluconic acid Filamentous Aspergillus niger fungi Organic acids Itaconicacid Filamentous Aspergillus terreus fungi Organic acids Itaconic acidFilamentous Aspergillus niger fungi Organic acids LCDAs - DDDA YeastCandida Organic acids Kojic Acid Filamentous Aspergillus oryzae fungiOrganic acids Kojic Acid Filamentous Aspergillus flavus fungi Organicacids Kojic Acid Filamentous Aspergillus tamarii fungi Organic acidsMalic Acid Filamentous Aspergillus oryzae fungi Organic acids Oxalicacid Filamentous Aspergillus niger fungi Organic acids Succinic acidFilamentous Aspergillus saccarolyticus fungi Organic acids Lactic acidFilamentous Aspergillus niger fungi Organic acids Lactic acidFilamentous Aspergillus brasiliensis fungi Hypolipidemic agentLovastatin Filamentous Aspergillus terreus fungi Melanogenesis inhibitorTerrein Filamentous Aspergillus terreus fungi Immunosuppresent drugCyclosporine A Filamentous Aspergillus terreus fungi Antiproliferativeagent Asperfuranone Filamentous Aspergillus terreus fungiAntiproliferative agent Asperfuranone Filamentous Aspergillus nidulansfungi Cholesterol-lowering Pyripyropene Filamentous Aspergillusfumigatus agent fungi Antibiotics Penicillin Filamentous Aspergillusoryzae fungi Antibiotics Penicillin Filamentous Aspergillus nidulansfungi Antimicrobial agent Fumagillin Filamentous Aspergillus fumigatusfungi Anticancer agent Fumitremorgin C Filamentous Aspergillus fumigatusfungi Anticancer agent Spirotryprostatins Filamentous Aspergillusfumigatus fungi Anticancer agent; Plinabulin Filamentous Aspergillusustus Antimicrobial agent fungi Anticancer agent PhenylahistinFilamentous Aspergillus ustus fungi Anticancer agent Stephacidin A & BFilamentous Aspergillus ochraceus fungi Anti cancer agent AsperphenamateFilamentous Aspergillus flavus fungi Cholecystokinin AsperlicinFilamentous Aspergillus alliaceus antagonist fungi Industrial enzymeAlpha-amylase Filamentous Aspergillus niger fungi Industrial enzymeAlpha-amylase Filamentous Aspergillus oryzae fungi Industrial enzymeAminopeptidase Filamentous Aspergillus niger fungi Industrial enzymeAminopeptidase Filamentous Aspergillus oryzae fungi Industrial enzymeAminopeptidase Filamentous Aspergillus sojae fungi Industrial enzyme AMPdeaminase Filamentous Aspergillus melleus fungi Industrial enzymeCatalase Filamentous Aspergillus niger fungi Industrial enzyme CellulaseFilamentous Aspergillus niger fungi Industrial enzyme ChymosinFilamentous Aspergillus niger fungi Industrial enzyme EsteraseFilamentous Aspergillus niger fungi Industrial enzyme Alpha- FilamentousAspergillus niger galactosidase fungi Industrial enzyme Beta-glucanaseFilamentous Aspergillus niger fungi Industrial enzyme Beta-glucanaseFilamentous Aspergillus aculeatus fungi Industrial enzyme Glucoseoxidase Filamentous Aspergillus niger fungi Industrial enzymeGlutaminase Filamentous Aspergillus oryzae fungi Industrial enzymeGlutaminase Filamentous Aspergillus sojae fungi Industrial enzymeBeta-D- Filamentous Aspergillus niger Glucosidase fungi Industrialenzyme Inulinase Filamentous Aspergillus niger fungi Industrial enzymeLactase Filamentous Aspergillus niger fungi Industrial enzyme LipaseFilamentous Aspergillus niger fungi Industrial enzyme Lipase FilamentousAspergillus oryzae fungi Industrial enzyme Xylanase FilamentousAspergillus niger fungiSelection Criteria and Goals

The selection criteria applied to the methods of the present disclosurewill vary with the specific goals of the strain improvement program. Thepresent disclosure may be adapted to meet any program goals. Forexample, in some embodiments, the program goal may be to maximize singlebatch yields of reactions with no immediate time limits. In otherembodiments, the program goal may be to rebalance biosynthetic yields toproduce a specific product, or to produce a particular ratio ofproducts. In other embodiments, the program goal may be to modify thechemical structure of a product, such as lengthening the carbon chain ofa polymer. In some embodiments, the program goal may be to improveperformance characteristics such as yield, titer, productivity,by-product elimination, tolerance to process excursions, optimal growthtemperature and growth rate. In some embodiments, the program goal isimproved host performance as measured by volumetric productivity,specific productivity, yield or titre, of a product of interest producedby a microbe.

In other embodiments, the program goal may be to optimize synthesisefficiency of a commercial strain in terms of final product yield perquantity of inputs (e.g., total amount of ethanol produced per pound ofsucrose). In other embodiments, the program goal may be to optimizesynthesis speed, as measured for example in terms of batch completionrates, or yield rates in continuous culturing systems. In otherembodiments, the program goal may be to increase strain resistance to aparticular phage, or otherwise increase strain vigor/robustness underculture conditions.

In some embodiments, strain improvement projects may be subject to morethan one goal. In some embodiments, the goal of the strain project mayhinge on quality, reliability, or overall profitability. In someembodiments, the present disclosure teaches methods of associatedselected mutations or groups of mutations with one or more of the strainproperties described above.

Persons having ordinary skill in the art will recognize how to tailorstrain selection criteria to meet the particular project goal. Forexample, selections of a strain's single batch max yield at reactionsaturation may be appropriate for identifying strains with high singlebatch yields. Selection based on consistency in yield across a range oftemperatures and conditions may be appropriate for identifying strainswith increased robustness and reliability.

In some embodiments, the selection criteria for the initialhigh-throughput phase and the tank-based validation will be identical.In other embodiments, tank-based selection may operate under additionaland/or different selection criteria. For example, in some embodiments,high-throughput strain selection might be based on single batch reactioncompletion yields, while tank-based selection may be expanded to includeselections based on yields for reaction speed.

Sequencing

In some embodiments, the present disclosure teaches whole-genomesequencing of the organisms described herein. In other embodiments, thepresent disclosure also teaches sequencing of plasmids, PCR products,and other oligos as quality controls to the methods of the presentdisclosure. Sequencing methods for large and small projects are wellknown to those in the art.

In some embodiments, any high-throughput technique for sequencingnucleic acids can be used in the methods of the disclosure. In someembodiments, the present disclosure teaches whole genome sequencing. Inother embodiments, the present disclosure teaches amplicon sequencingultra deep sequencing to identify genetic variations. In someembodiments, the present disclosure also teaches novel methods forlibrary preparation, including tagmentation (see WO/2016/073690). DNAsequencing techniques include classic dideoxy sequencing reactions(Sanger method) using labeled terminators or primers and gel separationin slab or capillary; sequencing by synthesis using reversiblyterminated labeled nucleotides, pyrosequencing; 454 sequencing; allelespecific hybridization to a library of labeled oligonucleotide probes;sequencing by synthesis using allele specific hybridization to a libraryof labeled clones that is followed by ligation; real time monitoring ofthe incorporation of labeled nucleotides during a polymerization step;polony sequencing; and SOLiD sequencing.

In one aspect of the disclosure, high-throughput methods of sequencingare employed that comprise a step of spatially isolating individualmolecules on a solid surface where they are sequenced in parallel. Suchsolid surfaces may include nonporous surfaces (such as in Solexasequencing, e.g. Bentley et al, Nature, 456: 53-59 (2008) or CompleteGenomics sequencing, e.g. Drmanac et al, Science, 327: 78-81 (2010)),arrays of wells, which may include bead- or particle-bound templates(such as with 454, e.g. Margulies et al, Nature, 437: 376-380 (2005) orIon Torrent sequencing, U.S. patent publication 2010/0137143 or2010/0304982), micromachined membranes (such as with SMRT sequencing,e.g. Eid et al, Science, 323: 133-138 (2009)), or bead arrays (as withSOLiD sequencing or polony sequencing, e.g. Kim et al, Science, 316:1481-1414 (2007)).

In another embodiment, the methods of the present disclosure compriseamplifying the isolated molecules either before or after they arespatially isolated on a solid surface. Prior amplification may compriseemulsion-based amplification, such as emulsion PCR, or rolling circleamplification. Also taught is Solexa-based sequencing where individualtemplate molecules are spatially isolated on a solid surface, afterwhich they are amplified in parallel by bridge PCR to form separateclonal populations, or clusters, and then sequenced, as described inBentley et al (cited above) and in manufacturer's instructions (e.g.TruSeq™ Sample Preparation Kit and Data Sheet, Illumina, Inc., SanDiego, Calif., 2010); and further in the following references: U.S. Pat.Nos. 6,090,592; 6,300,070; 7,115,400; and EP0972081B1; which areincorporated by reference.

In one embodiment, individual molecules disposed and amplified on asolid surface form clusters in a density of at least 10⁵ clusters percm²; or in a density of at least 5×10⁵ per cm²; or in a density of atleast 10⁶ clusters per cm². In one embodiment, sequencing chemistriesare employed having relatively high error rates. In such embodiments,the average quality scores produced by such chemistries aremonotonically declining functions of sequence read lengths. In oneembodiment, such decline corresponds to 0.5 percent of sequence readshave at least one error in positions 1-75; 1 percent of sequence readshave at least one error in positions 76-100; and 2 percent of sequencereads have at least one error in positions 101-125.

Computational Analysis and Prediction of Effects of Genome-Wide GeneticDesign Criteria

In some embodiments, the present disclosure teaches methods ofpredicting the effects of particular genetic alterations beingincorporated into a given host strain. In further aspects, thedisclosure provides methods for generating proposed genetic alterationsthat should be incorporated into a given host strain, in order for saidhost to possess a particular phenotypic trait or strain parameter. Ingiven aspects, the disclosure provides predictive models that can beutilized to design novel host strains. The novel host strains can befilamentous fungal host strains such as for example A. niger.

In some embodiments, the present disclosure teaches methods of analyzingthe performance results of each round of screening and methods forgenerating new proposed genome-wide sequence modifications predicted toenhance strain performance in the following round of screening.

In some embodiments, the present disclosure teaches that the systemgenerates proposed sequence modifications to host strains based onprevious screening results. In some embodiments, the recommendations ofthe present system are based on the results from the immediatelypreceding screening. In other embodiments, the recommendations of thepresent system are based on the cumulative results of one or more of thepreceding screenings.

In some embodiments, the recommendations of the present system are basedon previously developed HTP genetic design libraries. For example, insome embodiments, the present system is designed to save results fromprevious screenings, and apply those results to a different project, inthe same or different host organisms.

In other embodiments, the recommendations of the present system arebased on scientific insights. For example, in some embodiments, therecommendations are based on known properties of genes (from sourcessuch as annotated gene databases and the relevant literature), codonoptimization, transcriptional slippage, uORFs, or other hypothesisdriven sequence and host optimizations.

In some embodiments, the proposed sequence modifications to a hoststrain recommended by the system, or predictive model, are carried outby the utilization of one or more of the disclosed molecular tools setscomprising: (1) Promoter swaps, (2) SNP swaps, (3) Start/Stop codonexchanges, (4) Sequence optimization, (5) Stop swaps, and (5) Epistasismapping.

The HTP genetic engineering platform described herein is agnostic withrespect to any particular microbe or phenotypic trait (e.g. productionof a particular compound). That is, the platform and methods taughtherein can be utilized with any host cell to engineer said host cell tohave any desired phenotypic trait. Furthermore, the lessons learned froma given HTP genetic engineering process used to create one novel hostcell, can be applied to any number of other host cells, as a result ofthe storage, characterization, and analysis of a myriad of processparameters that occurs during the taught methods. In particular aspects,the host cells can be any coenocytic organism known in the art. Forexample, the host cell can be a filamentous fungal host cell. An exampleof a filamentous fungal host cell for use herein can be A. niger.

As alluded to in the epistatic mapping section, it is possible toestimate the performance (a.k.a. score) of a hypothetical strainobtained by consolidating a collection of mutations from a HTP geneticdesign library into a particular background via some preferredpredictive model. Given such a predictive model, it is possible to scoreand rank all hypothetical strains accessible to the mutation library viacombinatorial consolidation. The below section outlines particularmodels utilized in the present HTP platform.

Predictive Strain Design

Described herein is an approach for predictive strain design, including:methods of describing genetic changes and strain performance, predictingstrain performance based on the composition of changes in the strain,recommending candidate designs with high predicted performance, andfiltering predictions to optimize for second-order considerations, e.g.similarity to existing strains, epistasis, or confidence in predictions.

Inputs to Strain Design Model

In one embodiment, for the sake of ease of illustration, input data maycomprise two components: (1) sets of genetic changes and (2) relativestrain performance. Those skilled in the art will recognize that thismodel can be readily extended to consider a wide variety of inputs,while keeping in mind the countervailing consideration of overfitting.In addition to genetic changes, some of the input parameters(independent variables) that can be adjusted are cell types (genus,species, strain, phylogenetic characterization, etc.) and processparameters (e.g., environmental conditions, handling equipment,modification techniques, etc.) under which fermentation is conductedwith the cells.

The sets of genetic changes can come from the previously discussedcollections of genetic perturbations termed HTP genetic designlibraries. The relative strain performance can be assessed based uponany given parameter or phenotypic trait of interest (e.g. production ofa compound, small molecule, or product of interest).

Cell types can be specified in general categories such as prokaryoticand eukaryotic systems, genus, species, strain, tissue cultures (vs.disperse cells), etc. Process parameters that can be adjusted includetemperature, pressure, reactor configuration, and medium composition.Examples of reactor configuration include the volume of the reactor,whether the process is a batch or continuous, and, if continuous, thevolumetric flow rate, etc. One can also specify the support structure,if any, on which the cells reside. Examples of medium compositioninclude the concentrations of electrolytes, nutrients, waste products,acids, pH, and the like.

Sets of Genetic Changes from Selected HTP Genetic Design Libraries to beUtilized in the Initial Linear Regression Model that Subsequently isUsed to Create the Predictive Strain Design Model

To create a predictive strain design model, genetic changes in strainsof the same microbial species are first selected. The history of eachgenetic change is also provided (e.g., showing the most recentmodification in this strain lineage—“last change”). Thus, comparing thisstrain's performance to the performance of its parent represents a datapoint concerning the performance of the “last change” mutation.

Built Strain Performance Assessment

The goal of the taught model is to predict strain performance based onthe composition of genetic changes introduced to the strain. Toconstruct a standard for comparison, strain performance is computedrelative to a common reference strain, by first calculating the medianperformance per strain, per assay plate. Relative performance is thencomputed as the difference in average performance between an engineeredstrain and the common reference strain within the same plate.Restricting the calculations to within-plate comparisons ensures thatthe samples under consideration all received the same experimentalconditions.

FIG. 41 shows a hypothetic example in which the distribution of relativestrain performances for the input data is under consideration. Arelative performance of zero indicates that the engineered strainperformed equally well to the in-plate base or “reference” strain. Ofinterest is the ability of the predictive model to identify the strainsthat are likely to perform significantly above zero. Further, and moregenerally, of interest is whether any given strain outperforms itsparent by some criteria. In practice, the criteria can be a producttiter meeting or exceeding some threshold above the parent level, thoughhaving a statistically significant difference from the parent in thedesired direction could also be used instead or in addition. The role ofthe base or “reference” strain is simply to serve as an addednormalization factor for making comparisons within or between plates.

A concept to keep in mind is that of differences between: parent strainand reference strain. The parent strain is the background that was usedfor a current round of mutagenesis. The reference strain is a controlstrain run in every plate to facilitate comparisons, especially betweenplates, and is typically the “base strain” as referenced above. Butsince the base strain (e.g., the wild-type or industrial strain beingused to benchmark overall performance) is not necessarily a “base” inthe sense of being a mutagenesis target in a given round of strainimprovement, a more descriptive term is “reference strain.”

In summary, a base/reference strain is used to benchmark the performanceof built strains, generally, while the parent strain is used tobenchmark the performance of a specific genetic change in the relevantgenetic background.

Ranking the Performance of Built Strains with Linear Regression

The goal of the disclosed model is to rank the performance of builtstrains, by describing relative strain performance, as a function of thecomposition of genetic changes introduced into the built strains. Asdiscussed throughout the disclosure, the various HTP genetic designlibraries provide the repertoire of possible genetic changes (e.g.,genetic perturbations/alterations) that are introduced into theengineered strains. Linear regression is the basis for the currentlydescribed exemplary predictive model.

Genetic changes and their effect on relative performance is then inputfor regression-based modeling. The strain performances are rankedrelative to a common base strain, as a function of the composition ofthe genetic changes contained in the strain.

Linear Regression to Characterize Built Strains

Linear regression is an attractive method for the described HTP genomicengineering platform, because of the ease of implementation andinterpretation. The resulting regression coefficients can be interpretedas the average increase or decrease in relative strain performanceattributable to the presence of each genetic change.

For example, in some embodiments, this technique allows us to concludethat changing the original promoter to another promoter improvesrelative strain performance by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more units on average and is thus a potentially highly desirablechange, in the absence of any negative epistatic interactions (note: theinput is a unit-less normalized value).

The taught method therefore uses linear regression models todescribe/characterize and rank built strains, which have various geneticperturbations introduced into their genomes from the various taughtlibraries.

Predictive Design Modeling

The linear regression model described above, which utilized data fromconstructed strains, can be used to make performance predictions forstrains that haven't yet been built.

The procedure can be summarized as follows: generate in silico allpossible configurations of genetic changes→use the regression model topredict relative strain performance→order the candidate strain designsby performance. Thus, by utilizing the regression model to predict theperformance of as-yet-unbuilt strains, the method allows for theproduction of higher performing strains, while simultaneously conductingfewer experiments.

Generate Configurations

When constructing a model to predict performance of as-yet-unbuiltstrains, the first step is to produce a sequence of design candidates.This is done by fixing the total number of genetic changes in thestrain, and then defining all possible combinations of genetic changes.For example, one can set the total number of potential geneticchanges/perturbations to 29 (e.g. 29 possible SNPs, or 29 differentpromoters, or any combination thereof as long as the universe of geneticperturbations is 29) and then decide to design all possible 3-membercombinations of the 29 potential genetic changes, which will result in3,654 candidate strain designs.

To provide context to the aforementioned 3,654 candidate strains,consider that one can calculate the number of non-redundant groupings ofsize r from n possible members using: n!/((n−r)!*r!). If r=3, n=29 gives3,654. Thus, if one designs all possible 3-member combinations of 29potential changes the results is 3,654 candidate strains.

Predict Performance of New Strain Designs

Using the linear regression constructed above with the combinatorialconfigurations as input, one can then predict the expected relativeperformance of each candidate design. For example, the composition ofchanges for the top 100 predicted strain designs can be summarized in a2-dimensional map, in which the x-axis lists the pool of potentialgenetic changes (29 possible genetic changes), and the y-axis shows therank order.

Predictive accuracy should increase over time as new observations areused to iteratively retrain and refit the model. Results from a study bythe inventors illustrate the methods by which the predictive model canbe iteratively retrained and improved. The quality of model predictionscan be assessed through several methods, including a correlationcoefficient indicating the strength of association between the predictedand observed values, or the root-mean-square error, which is a measureof the average model error. Using a chosen metric for model evaluation,the system may define rules for when the model should be retrained.

A couple of unstated assumptions to the above model include: (1) thereare no epistatic interactions; and (2) the genetic changes/perturbationsutilized to build the predictive model were all made in the samebackground, as the proposed combinations of genetic changes.

Filtering for Second-Order Features

The above illustrative example focused on linear regression predictionsbased on predicted host cell performance. In some embodiments, thepresent linear regression methods can also be applied to non-biomoleculefactors, such as saturation biomass, resistance, or other measurablehost cell features. Thus, the methods of the present disclosure alsoteach considering other features outside of predicted performance whenprioritizing the candidates to build. Assuming there is additionalrelevant data, nonlinear terms are also included in the regressionmodel.

Closeness with Existing Strains

Predicted strains that are similar to ones that have already been builtcould result in time and cost savings despite not being a top predictedcandidate

Diversity of Changes

When constructing the aforementioned models, one cannot be certain thatgenetic changes will truly be additive (as assumed by linear regressionand mentioned as an assumption above) due to the presence of epistaticinteractions. Therefore, knowledge of genetic change dissimilarity canbe used to increase the likelihood of positive additivity. If one knows,for example, that the changes from the top ranked strain are on the samemetabolic pathway and have similar performance characteristics, thenthat information could be used to select another top ranking strain witha dissimilar composition of changes. As described in the section aboveconcerning epistasis mapping, the predicted best genetic changes may befiltered to restrict selection to mutations with sufficiently dissimilarresponse profiles. Alternatively, the linear regression may be aweighted least squares regression using the similarity matrix to weightpredictions.

Diversity of Predicted Performance

Finally, one may choose to design strains with middling or poorpredicted performance, in order to validate and subsequently improve thepredictive models.

Iterative Strain Design Optimization

In embodiments, the order placement engine 208 places a factory order tothe factory 210 to manufacture microbial strains incorporating the topcandidate mutations. In feedback-loop fashion, the results may beanalyzed by the analysis equipment 214 to determine which microbesexhibit desired phenotypic properties (314). During the analysis phase,the modified strain cultures are evaluated to determine theirperformance, i.e., their expression of desired phenotypic properties,including the ability to be produced at industrial scale. For example,the analysis phase uses, among other things, image data of plates tomeasure microbial colony growth as an indicator of colony health. Theanalysis equipment 214 is used to correlate genetic changes withphenotypic performance, and save the resulting genotype-phenotypecorrelation data in libraries, which may be stored in library 206, toinform future microbial production.

In particular, the candidate changes that actually result insufficiently high measured performance may be added as rows in thedatabase to tables. In this manner, the best performing mutations areadded to the predictive strain design model in a supervised machinelearning fashion.

LIMS iterates the design/build/test/analyze cycle based on thecorrelations developed from previous factory runs. During a subsequentcycle, the analysis equipment 214 alone, or in conjunction with humanoperators, may select the best candidates as base strains for input backinto input interface 202, using the correlation data to fine tunegenetic modifications to achieve better phenotypic performance withfiner granularity. In this manner, the laboratory information managementsystem of embodiments of the disclosure implements a quality improvementfeedback loop.

In sum, with reference to the flowchart of FIG. 16 the iterativepredictive strain design workflow may be described as follows:

Generate a training set of input and output variables, e.g., geneticchanges as inputs and performance features as outputs (3302). Generationmay be performed by the analysis equipment 214 based upon previousgenetic changes and the corresponding measured performance of themicrobial strains incorporating those genetic changes.

Develop an initial model (e.g., linear regression model) based upontraining set (3304). This may be performed by the analysis equipment214.

Generate design candidate strains (3306)

In one embodiment, the analysis equipment 214 may fix the number ofgenetic changes to be made to a background strain, in the form ofcombinations of changes. To represent these changes, the analysisequipment 214 may provide to the interpreter 204 one or more DNAspecification expressions representing those combinations of changes.(These genetic changes or the microbial strains incorporating thosechanges may be referred to as “test inputs.”) The interpreter 204interprets the one or more DNA specifications, and the execution engine207 executes the DNA specifications to populate the DNA specificationwith resolved outputs representing the individual candidate designstrains for those changes.

Based upon the model, the analysis equipment 214 predicts expectedperformance of each candidate design strain (3308).

The analysis equipment 214 selects a limited number of candidatedesigns, e.g., 100, with highest predicted performance (3310).

As described elsewhere herein with respect to epistasis mapping, theanalysis equipment 214 may account for second-order effects such asepistasis, by, e.g., filtering top designs for epistatic effects, orfactoring epistasis into the predictive model.

Build the filtered candidate strains (at the factory 210) based on thefactory order generated by the order placement engine 208 (3312).

The analysis equipment 214 measures the actual performance of theselected strains, selects a limited number of those selected strainsbased upon their superior actual performance (3314), and adds the designchanges and their resulting performance to the predictive model (3316).In the linear regression example, add the sets of design changes andtheir associated performance as new rows in a table.

The analysis equipment 214 then iterates back to generation of newdesign candidate strains (3306), and continues iterating until a stopcondition is satisfied. The stop condition may comprise, for example,the measured performance of at least one microbial strain satisfying aperformance metric, such as yield, growth rate, or titer.

In the example above, the iterative optimization of strain designemploys feedback and linear regression to implement machine learning. Ingeneral, machine learning may be described as the optimization ofperformance criteria, e.g., parameters, techniques or other features, inthe performance of an informational task (such as classification orregression) using a limited number of examples of labeled data, and thenperforming the same task on unknown data. In supervised machine learningsuch as that of the linear regression example above, the machine (e.g.,a computing device) learns, for example, by identifying patterns,categories, statistical relationships, or other attributes, exhibited bytraining data. The result of the learning is then used to predictwhether new data will exhibit the same patterns, categories, statisticalrelationships or other attributes.

Embodiments of the disclosure may employ other supervised machinelearning techniques when training data is available. In the absence oftraining data, embodiments may employ unsupervised machine learning.Alternatively, embodiments may employ semi-supervised machine learning,using a small amount of labeled data and a large amount of unlabeleddata. Embodiments may also employ feature selection to select the subsetof the most relevant features to optimize performance of the machinelearning model. Depending upon the type of machine learning approachselected, as alternatives or in addition to linear regression,embodiments may employ for example, logistic regression, neuralnetworks, support vector machines (SVMs), decision trees, hidden Markovmodels, Bayesian networks, Gram Schmidt, reinforcement-based learning,cluster-based learning including hierarchical clustering, geneticalgorithms, and any other suitable learning machines known in the art.In particular, embodiments may employ logistic regression to provideprobabilities of classification (e.g., classification of genes intodifferent functional groups) along with the classifications themselves.See, e.g., Shevade, A simple and efficient algorithm for gene selectionusing sparse logistic regression, Bioinformatics, Vol. 19, No. 17 2003,pp. 2246-2253, Leng, et al., Classification using functional dataanalysis for temporal gene expression data, Bioinformatics, Vol. 22, No.1, Oxford University Press (2006), pp. 68-76, all of which areincorporated by reference in their entirety herein.

Embodiments may employ graphics processing unit (GPU) acceleratedarchitectures that have found increasing popularity in performingmachine learning tasks, particularly in the form known as deep neuralnetworks (DNN). Embodiments of the disclosure may employ GPU-basedmachine learning, such as that described in GPU-Based Deep LearningInference: A Performance and Power Analysis, NVidia Whitepaper, November2015, Dahl, et al., Multi-task Neural Networks for QSAR Predictions,Dept. of Computer Science, Univ. of Toronto, June 2014 (arXiv:1406.1231[stat.ML]), all of which are incorporated by reference in their entiretyherein. Machine learning techniques applicable to embodiments of thedisclosure may also be found in, among other references, Libbrecht, etal., Machine learning applications in genetics and genomics, NatureReviews: Genetics, Vol. 16, June 2015, Kashyap, et al., Big DataAnalytics in Bioinformatics: A Machine Learning Perspective, Journal ofLatex Class Files, Vol. 13, No. 9, September 2014, Prompramote, et al.,Machine Learning in Bioinformatics, Chapter 5 of BioinformaticsTechnologies, pp. 117-153, Springer Berlin Heidelberg 2005, all of whichare incorporated by reference in their entirety herein.

Iterative Predictive Strain Design: Example

The following provides an example application of the iterativepredictive strain design workflow outlined above.

An initial set of training inputs and output variables was prepared.This set comprised 1864 unique engineered strains with defined geneticcomposition. Each strain contained between 5 and 15 engineered changes.A total of 336 unique genetic changes were present in the training.

An initial predictive computer model was developed. The implementationused a generalized linear model (Kernel Ridge Regression with 4th orderpolynomial kernel). The implementation models two distinct phenotypes(yield and productivity). These phenotypes were combined as weighted sumto obtain a single score for ranking, as shown below. Various modelparameters, e.g. regularization factor, were tuned via k-fold crossvalidation over the designated training data.

The implementation does not incorporate any explicit analysis ofinteraction effects as described in the Epistasis Mapping section above.However, as those skilled in the art would understand, the implementedgeneralized linear model may capture interaction effects implicitlythrough the second, third and fourth order terms of the kernel.

The model is trained against the training set After training, asignificant quality fitting of the yield model to the training data canbe demonstrated.

Candidate strains are then generated. This embodiments includes a serialbuild constraint associated with the introduction of new genetic changesto a parent strain. Here, candidates are not considered simply as afunction of the desired number of changes. Instead, the analysisequipment 214 selects, as a starting point, a collection of previouslydesigned strains known to have high performance metrics (“seedstrains”). The analysis equipment 214 individually applies geneticchanges to each of the seed strains. The introduced genetic changes donot include those already present in the seed strain. For varioustechnical, biological or other reasons, certain mutations are explicitlyrequired, or explicitly excluded

Based upon the model, the analysis equipment 214 predicted theperformance of candidate strain designs. The analysis equipment 214ranks candidates from “best” to “worst” based on predicted performancewith respect to two phenotypes of interest (yield and productivity).Specifically, the analysis equipment 214 used a weighted sum to score acandidate strain:Score=0.8*yield/max(yields)+0.2*prod/max(prods),where yield represents predicted yield for the candidate strain,max(yields) represents the maximum yield over all candidate strains,prod represents productivity for the candidate strain, andmax(prods) represents the maximum yield over all candidate strains.

The analysis equipment 214 generates a final set of recommendations fromthe ranked list of candidates by imposing both capacity constraints andoperational constraints. In some embodiments, the capacity limit can beset at a given number, such as 48 computer-generated candidate designstrains.

The trained model (described above) can be used to predict the expectedperformance (for yield and productivity) of each candidate strain. Theanalysis equipment 214 can rank the candidate strains using the scoringfunction given above. Capacity and operational constraints can be thenapplied to yield a filtered set of 48 candidate strains. Filteredcandidate strains are then built (at the factory 210) based on a factoryorder generated by the order placement engine 208 (3312). The order canbe based upon DNA specifications corresponding to the candidate strains.

In practice, the build process has an expected failure rate whereby arandom set of strains is not built.

The analysis equipment 214 can also be used to measure the actual yieldand productivity performance of the selected strains. The analysisequipment 214 can evaluate the model and recommended strains based onthree criteria: model accuracy; improvement in strain performance; andequivalence (or improvement) to human expert-generated designs.

The yield and productivity phenotypes can be measured for recommendedstrains and compared to the values predicted by the model.

Next, the analysis equipment 214 computes percentage performance changefrom the parent strain for each of the recommended strains.

Predictive accuracy can be assessed through several methods, including acorrelation coefficient indicating the strength of association betweenthe predicted and observed values, or the root-mean-square error, whichis a measure of the average model error. Over many rounds ofexperimentation, model predictions may drift, and new genetic changesmay be added to the training inputs to improve predictive accuracy. Forthis example, design changes and their resulting performance were addedto the predictive model (3316).

Genomic Design and Engineering as a Service

In embodiments of the disclosure, the LIMS system software 3210 of FIG.15 may be implemented in a cloud computing system 3202 of FIG. 15, toenable multiple users to design and build microbial strains according toembodiments of the present disclosure. FIG. 15 illustrates a cloudcomputing environment 3204 according to embodiments of the presentdisclosure. Client computers 3206, such as those illustrated in FIG. 15,access the LIMS system via a network 3208, such as the Internet. Inembodiments, the LIMS system application software 3210 resides in thecloud computing system 3202. The LIMS system may employ one or morecomputing systems using one or more processors, of the type illustratedin FIG. 15. The cloud computing system itself includes a networkinterface 3212 to interface the LIMS system applications 3210 to theclient computers 3206 via the network 3208. The network interface 3212may include an application programming interface (API) to enable clientapplications at the client computers 3206 to access the LIMS systemsoftware 3210. In particular, through the API, client computers 3206 mayaccess components of the LIMS system 200, including without limitationthe software running the input interface 202, the interpreter 204, theexecution engine 207, the order placement engine 208, the factory 210,as well as test equipment 212 and analysis equipment 214. A software asa service (SaaS) software module 3214 offers the LIMS system software3210 as a service to the client computers 3206. A cloud managementmodule 3216 manages access to the LIMS system 3210 by the clientcomputers 3206. The cloud management module 3216 may enable a cloudarchitecture that employs multitenant applications, virtualization orother architectures known in the art to serve multiple users. FIG. 44depicts a proof of principle of the utility of the LIMS system asapplied to a filamentous fungal host cell system.

Genomic Automation

Automation of the methods of the present disclosure enableshigh-throughput phenotypic screening and identification of targetproducts from multiple test strain variants simultaneously.

The aforementioned genomic engineering predictive modeling platform ispremised upon the fact that hundreds and thousands of mutant strains areconstructed in a high-throughput fashion. The robotic and computersystems described below are the structural mechanisms by which such ahigh-throughput process can be carried out.

In some embodiments, the present disclosure teaches methods of improvinghost cell productivities, or rehabilitating industrial strains. As partof this process, the present disclosure teaches methods of assemblingDNA, building new strains, screening cultures in plates, and screeningcultures in models for tank fermentation. In some embodiments, thepresent disclosure teaches that one or more of the aforementionedmethods of creating and testing new host strains is aided by automatedrobotics.

In some embodiments, the present disclosure teaches a high-throughputstrain engineering platform as depicted in FIG. 6A-B.

HTP Robotic System

In some embodiments, the methods and systems provided herein compriseautomated steps. For example, the generation of protoplasts,transformation of protoplasts, screening transformed protoplasts by NGSprior to purification, purifying homokaryotic protoplasts viaselection/counterselection and screening transformed protoplasts by NGSafter purification as described herein can be automated. As describedherein, the methods and system can contain a further step of screeningpurified homokaryotic transformants for the production of a protein ormetabolite of interest. The automated methods of the disclosure cancomprise a robotic system. The systems outlined herein can be generallydirected to the use of 96- or 384-well microtiter plates, but as will beappreciated by those in the art, any number of different plates orconfigurations may be used. In addition, any or all of the stepsoutlined herein may be automated; thus, for example, the systems may becompletely or partially automated. The automated methods and systems canbe high-throughput. For purposes of this disclosure, high-throughputscreening can refers to any partially- or fully-automated method that iscapable of evaluating about 1,000 or more transformants per day, andparticularly to those methods capable of evaluating 5,000 or moretransformants per day, and most particularly to methods capable ofevaluating 10,000 or more transformants per day. The partially orfully-automated methods can entail the use of one or more liquidhandling steps.

As described herein, the methods and system provided herein can comprisea screening step such that a transformant generated and purified asdescribed herein is screened or tested for the production of a productof interest. The product of interest can be any product of interestprovided herein such as, for example, an alcohol, pharmaceutical,metabolite, protein, enzyme, amino acid, or acid (e.g., citric acid).Accordingly, the methods and systems provided herein can furthercomprise culturing a clonal colony or culture purified according to themethods of the disclosure, under conditions permitting expression andsecretion of the product of interest and recovering the subsequentlyproduced product of interest. As described herein, the product ofinterest can an exogenous and/or heterologous protein or a metaboliteproduced as the result of the expression of an exogenous and orheterologous protein.

In some embodiments, the automated methods of the disclosure comprise arobotic system. The systems outlined herein are generally directed tothe use of 96- or 384-well microtiter plates, but as will be appreciatedby those in the art, any number of different plates or configurationsmay be used. In addition, any or all of the steps outlined herein may beautomated; thus, for example, the systems may be completely or partiallyautomated.

In some embodiments, the automated systems of the present disclosurecomprise one or more work modules. For example, in some embodiments, theautomated system of the present disclosure comprises a DNA synthesismodule, a vector cloning module, a strain transformation module, ascreening module, and a sequencing module (see FIG. 7).

As will be appreciated by those in the art, an automated system caninclude a wide variety of components, including, but not limited to:liquid handlers; one or more robotic arms; plate handlers for thepositioning of microplates; plate sealers, plate piercers, automated lidhandlers to remove and replace lids for wells on non-cross contaminationplates; disposable tip assemblies for sample distribution withdisposable tips; washable tip assemblies for sample distribution; 96well loading blocks; integrated thermal cyclers; cooled reagent racks;microtiter plate pipette positions (optionally cooled); stacking towersfor plates and tips; magnetic bead processing stations; filtrationssystems; plate shakers; barcode readers and applicators; and computersystems. FIG. 8 depicts an overview of an integrated filamentous fungalstrain improvement program of the present disclosure.

In some embodiments, the robotic systems of the present disclosureinclude automated liquid and particle handling enabling high-throughputpipetting to perform all the steps in the process of gene targeting andrecombination applications. This includes liquid and particlemanipulations such as aspiration, dispensing, mixing, diluting, washing,accurate volumetric transfers; retrieving and discarding of pipettetips; and repetitive pipetting of identical volumes for multipledeliveries from a single sample aspiration. These manipulations arecross-contamination-free liquid, particle, cell, and organism transfers.The instruments perform automated replication of microplate samples tofilters, membranes, and/or daughter plates, high-density transfers,full-plate serial dilutions, and high capacity operation.

The automated system can be any known automated high-throughput systemknown in the art. For example, the automated system can be the automatedmicroorganism handling tool is described in Japanese patent applicationpublication number 11-304666. This device is capable of the transfer ofmicrodroplets containing individual cells, and it is anticipated thatthe fungal strains of the present disclosure, by virtue of theirmorphology, will be amenable to micromanipulation of individual cloneswith this device. An additional example of an automated system for usein the methods and system of the present disclosure is the automatedmicrobiological high-throughput screening system described in Beydon etal., J. Biomol. Screening 5:13 21 (2000). The automated system for useherein can be a customized automated liquid handling system. In someembodiments, the customized automated liquid handling system of thedisclosure is a TECAN machine (e.g. a customized TECAN Freedom Evo).

In some embodiments, the automated systems of the present disclosure arecompatible with platforms for multi-well plates, deep-well plates,square well plates, reagent troughs, test tubes, mini tubes, microfugetubes, cryovials, filters, micro array chips, optic fibers, beads,agarose and acrylamide gels, and other solid-phase matrices or platformsare accommodated on an upgradeable modular deck. In some embodiments,the automated systems of the present disclosure contain at least onemodular deck for multi-position work surfaces for placing source andoutput samples, reagents, sample and reagent dilution, assay plates,sample and reagent reservoirs, pipette tips, and an active tip-washingstation.

In some embodiments, the automated systems of the present disclosureinclude high-throughput electroporation systems. In some embodiments,the high-throughput electroporation systems are capable of transformingcells in 96 or 384-well plates. In some embodiments, the high-throughputelectroporation systems include VWR® High-throughput ElectroporationSystems, BTX™, Bio-Rad® Gene Pulser MXcel™ or other multi-wellelectroporation system.

In some embodiments, the integrated thermal cycler and/or thermalregulators are used for stabilizing the temperature of heat exchangerssuch as controlled blocks or platforms to provide accurate temperaturecontrol of incubating samples from 0° C. to 100° C.

In some embodiments, the automated systems of the present disclosure arecompatible with interchangeable machine-heads (single or multi-channel)with single or multiple magnetic probes, affinity probes, replicators orpipetters, capable of robotically manipulating liquid, particles, cells,and multi-cellular organisms. Multi-well or multi-tube magneticseparators and filtration stations manipulate liquid, particles, cells,and organisms in single or multiple sample formats.

In some embodiments, the automated systems of the present disclosure arecompatible with camera vision and/or spectrometer systems. Thus, in someembodiments, the automated systems of the present disclosure are capableof detecting and logging color and absorption changes in ongoingcellular cultures.

In some embodiments, the automated system of the present disclosure isdesigned to be flexible and adaptable with multiple hardware add-ons toallow the system to carry out multiple applications. The softwareprogram modules allow creation, modification, and running of methods.The system's diagnostic modules allow setup, instrument alignment, andmotor operations. The customized tools, labware, and liquid and particletransfer patterns allow different applications to be programmed andperformed. The database allows method and parameter storage. Robotic andcomputer interfaces allow communication between instruments.

Thus, in some embodiments, the present disclosure teaches ahigh-throughput strain engineering platform, as depicted in FIGS. 11 and12.

Persons having skill in the art will recognize the various roboticplatforms capable of carrying out the HTP engineering methods of thepresent disclosure. Table 3 below provides a non-exclusive list ofscientific equipment capable of carrying out each step of the HTPengineering steps of the present disclosure as described in FIGS. 11 and12.

TABLE 3 Non-exclusive list of Scientific Equipment Compatible with theHTP engineering methods of the present disclosure. Compatible EquipmentEquipment Type Operation(s) performed Make/Model/Configuration Acquireand build liquid handlers Hitpicking (combining Hamilton Microlab STAR,DNA pieces by transferring) Labcyte Echo 550, Tecan EVOprimers/templates for 200, Beckman Coulter Biomek PCR amplification ofFX, BioFluidix GmbH BioSpot DNA parts BT600 liquid handling workstation,or equivalents Thermal cyclers PCR amplification of Inheco Cycler, ABI2720, ABI DNA parts Proflex 384, ABI Veriti, or equivalents QC DNA partsFragment gel electrophoresis to Agilent Bioanalyzer, AATI analyzersconfirm PCR products of Fragment Analyzer, or (capillary appropriatesize equivalents electrophoresis) Sequencer Verifying sequence ofBeckman Ceq-8000, Beckman (sanger: parts/templates GenomeLab ™, orequivalents Beckman) NGS (next Verifying sequence of Illumina MiSeqseries sequences, generation parts/templates Illumina Hi-Seq, Iontorrent, pac sequencing) bio or other equivalents instrumentnanodrop/plate assessing concentration Molecular Devices SpectraMaxreader of DNA samples M5, Tecan M1000, or equivalents. Generate DNAliquid handlers Hitpicking (combining Hamilton Microlab STAR, assemblyby transferring) DNA Labcyte Echo 550, Tecan EVO parts for assemblyalong 200, Beckman Coulter Biomek with cloning vector, FX, BioFluidixGmbH BioSpot addition of reagents for BT600 liquid handling assemblyworkstation, or equivalents reaction/process QC DNA assembly Colonypickers for inoculating colonies Scirobotics Pickolo, Molecular inliquid media Devices QPix 420 liquid handlers Hitpicking HamiltonMicrolab STAR, primers/templates, Labcyte Echo 550, Tecan EVO dilutingsamples 200, Beckman Coulter Biomek FX, BioFluidix GmbH BioSpot BT600liquid handling workstation, or equivalents Fragment gel electrophoresisto Agilent Bioanalyzer, AATI analyzers confirm assembled FragmentAnalyzer (capillary products of appropriate electrophoresis) sizeSequencer Verifying sequence of ABI3730 Thermo Fisher, (sanger:assembled plasmids Beckman Ceq-8000, Beckman Beckman) GenomeLab ™, orequivalents NGS (next Verifying sequence of Illumina MiSeq seriessequences, generation assembled plasmids Illumina Hi-Seq, Ion torrent,pac sequencing) bio or other equivalents instrument Prepare base straincentrifuge spinning/pelleting cells Beckman Avanti floor centrifuge, andDNA assembly Hettich Centrifuge Transform DNA into Electroporatorselectroporative BTX Gemini X2, BIO-RAD base strain transformation ofcells MicroPulser Electroporator Ballistic ballistic transformationBIO-RAD PDS1000 transformation of cells Incubators, for chemical InhecoCycler, ABI 2720, ABI thermal cyclers transformation/heat Proflex 384,ABI Veriti, or shock equivalents Liquid handlers for combining DNA,Hamilton Microlab STAR, cells, buffer Labcyte Echo 550, Tecan EVO 200,Beckman Coulter Biomek FX, BioFluidix GmbH BioSpot BT600 liquid handlingworkstation, or equivalents Integrate DNA into Colony pickers forinoculating colonies Scirobotics Pickolo, Molecular genome of base inliquid media or Devices QPix 420 strain diluting spores Single fordispensing single Cellenion CellenONE, Berkeley cell/spore cells/sporesinto wells on Lights Beacon Instrument, FACS, dispensers microtiterplate or Cytena single cell printer Liquid handlers For transferringcells Hamilton Microlab STAR, onto Agar, transferring Labcyte Echo 550,Tecan EVO from culture plates to 200, Beckman Coulter Biomek differentculture plates FX, BioFluidix GmbH BioSpot (inoculation into other BT600liquid handling selective media) or workstation or equivalentsdispensing diluted spore preparations into microtiter plates Platformincubation with shaking Kuhner Shaker ISF4-X, Infors-ht shaker- ofmicrotiter plate Multitron Pro incubators cultures QC transformed Colonypickers for inoculating colonies Scirobotics Pickolo, Molecular strainin liquid media Devices QPix 420 liquid handlers Hitpicking HamiltonMicrolab STAR, primers/templates, Labcyte Echo 550, Tecan EVO dilutingsamples 200, Beckman Coulter Biomek FX, BioFluidix GmbH BioSpot BT600liquid handling workstation or equivalents Thermal cyclers cPCRverification of Inheco Cycler, ABI 2720, ABI strains Proflex 384, ABIVeriti, or equivalents Fragment gel electrophoresis to Infors-htMultitron Pro, Kuhner analyzers confirm cPCR products Shaker ISF4-X(capillary of appropriate size electrophoresis) Sequencer Sequenceverification of Beckman Ceq-8000, Beckman (sanger: introducedmodification GenomeLab ™, or equivalents Beckman) NGS (next Sequenceverification of Illumina MiSeq series sequences, generation introducedmodification Illumina Hi-Seq, Ion torrent, pac sequencing) bio or otherequivalents instrument Select and Liquid handlers For transferring fromHamilton Microlab STAR, consolidate culture plates to different LabcyteEcho 550, Tecan EVO QC' d strains culture plates 200, Beckman CoulterBiomek into test plate (inoculation into FX, BioFluidix GmbH BioSpotproduction media) BT600 liquid handling workstation or equivalentsColony pickers for inoculating colonies Scirobotics Pickolo, Molecularin liquid media Devices QPix 420 Platform incubation with shaking KuhnerShaker ISF4-X, Infors-ht shaker- of microtiter plate Multitron Proincubators cultures Culture strains Liquid handlers For transferringfrom Hamilton Microlab STAR, in seed plates culture plates to differentLabcyte Echo 550, Tecan EVO culture plates 200, Beckman Coulter Biomek(inoculation into FX, BioFluidix GmbH BioSpot production media) BT600liquid handling workstation or equivalents Platform incubation withshaking Kuhner Shaker ISF4-X, Infors-ht shaker- of microtiter plateMultitron Pro incubators cultures liquid Dispense liquid culture Wellmate (Thermo), Benchcel2R dispensers media into microtiter (velocity11), plateloc (velocity plates 11) microplate apply barcoders to platesMicroplate labeler (a2+ cab - labeler agilent), benchcell 6R(velocity11) Generate product Liquid handlers For transferring fromHamilton Microlab STAR, from strain culture plates to different LabcyteEcho 550, Tecan EVO culture plates 200, Beckman Coulter Biomek(inoculation into FX, BioFluidix GmbH BioSpot production media) BT600liquid handling workstation or equivalents Platform incubation withshaking Kuhner Shaker ISF4-X, Infors-ht shaker- of microtiter plateMultitron Pro incubators cultures liquid Dispense liquid culture wellmate (Thermo), Benchcel2R dispensers media into multiple (velocity 11),plateloc (velocity microtiter plates and seal 11) plates microplateApply barcodes to plates microplate labeler (a2+ cab - labeler agilent),benchcell 6R (velocity11) Evaluate Liquid handlers For processingculture Hamilton Microlab STAR, performance broth for downstream LabcyteEcho 550, Tecan EVO analytical 200, Beckman Coulter Biomek FX,BioFluidix GmbH BioSpot BT600 liquid handling workstation or equivalentsUHPLC, HPLC quantitative analysis of Agilent 1290 Series UHPLC andprecursor and target 1200 Series HPLC with UV and compounds RIdetectors, or equivalent; also any LC/MS LC/MS highly specific analysisAgilent 6490 QQQ and 6550 of precursor and target QTOF coupled to 1290Series compounds as well as UHPLC side and degradation productsSpectrophotometer Quantification of Tecan M1000, spectramax M5,different compounds Genesys 10S using spectrophotometer based assaysCulture strains in Fermenters: incubation with shaking Sartorius,DASGIPs (Eppendorf), flasks BIO-FLOs (Sartorius-stedim). ApplikonPlatform innova 4900, or any equivalent shakers Generate productFermenters: DASGIPs (Eppendorf), BIO-FLOs (Sartorius-stedim) from strainEvaluate performance Liquid handlers For transferring from HamiltonMicrolab STAR, culture plates to different Labcyte Echo 550, Tecan EVOculture plates 200, Beckman Coulter Biomek (inoculation into FX,BioFluidix GmbH BioSpot production media) BT600 liquid handlingworkstation or equivalents UHPLC, HPLC quantitative analysis of Agilent1290 Series UHPLC and precursor and target 1200 Series HPLC with UV andcompounds RI detectors, or equivalent; also any LC/MS LC/MS highlyspecific analysis Agilent 6490 QQQ and 6550 of precursor and target QTOFcoupled to 1290 Series compounds as well as UHPLC side and degradationproducts Flow cytometer Characterize strain BD Accuri, Millipore Guavaperformance (measure viability) Spectrophotometer Characterize strainTecan M1000, Spectramax M5, performance (measure or other equivalentsbiomass)Computer System Hardware

FIG. 17 illustrates an example of a computer system 800 that may be usedto execute program code stored in a non-transitory computer readablemedium (e.g., memory) in accordance with embodiments of the disclosure.The computer system includes an input/output subsystem 802, which may beused to interface with human users and/or other computer systemsdepending upon the application. The I/O subsystem 802 may include, e.g.,a keyboard, mouse, graphical user interface, touchscreen, or otherinterfaces for input, and, e.g., an LED or other flat screen display, orother interfaces for output, including application program interfaces(APIs). Other elements of embodiments of the disclosure, such as thecomponents of the LIMS system, may be implemented with a computer systemlike that of computer system 800.

Program code may be stored in non-transitory media such as persistentstorage in secondary memory 810 or main memory 808 or both. Main memory808 may include volatile memory such as random access memory (RAM) ornon-volatile memory such as read only memory (ROM), as well as differentlevels of cache memory for faster access to instructions and data.Secondary memory may include persistent storage such as solid statedrives, hard disk drives or optical disks. One or more processors 804reads program code from one or more non-transitory media and executesthe code to enable the computer system to accomplish the methodsperformed by the embodiments herein. Those skilled in the art willunderstand that the processor(s) may ingest source code, and interpretor compile the source code into machine code that is understandable atthe hardware gate level of the processor(s) 804. The processor(s) 804may include graphics processing units (GPUs) for handlingcomputationally intensive tasks. Particularly in machine learning, oneor more CPUs 804 may offload the processing of large quantities of datato one or more GPUs 804.

The processor(s) 804 may communicate with external networks via one ormore communications interfaces 807, such as a network interface card,WiFi transceiver, etc. A bus 805 communicatively couples the I/Osubsystem 802, the processor(s) 804, peripheral devices 806,communications interfaces 807, memory 808, and persistent storage 810.Embodiments of the disclosure are not limited to this representativearchitecture. Alternative embodiments may employ different arrangementsand types of components, e.g., separate buses for input-outputcomponents and memory subsystems.

Those skilled in the art will understand that some or all of theelements of embodiments of the disclosure, and their accompanyingoperations, may be implemented wholly or partially by one or morecomputer systems including one or more processors and one or more memorysystems like those of computer system 800. In particular, the elementsof the LIMS system 200 and any robotics and other automated systems ordevices described herein may be computer-implemented. Some elements andfunctionality may be implemented locally and others may be implementedin a distributed fashion over a network through different servers, e.g.,in client-server fashion, for example. In particular, server-sideoperations may be made available to multiple clients in a software as aservice (SaaS) fashion, as shown in FIG. 15.

The term component in this context refers broadly to software, hardware,or firmware (or any combination thereof) component. Components aretypically functional components that can generate useful data or otheroutput using specified input(s). A component may or may not beself-contained. An application program (also called an “application”)may include one or more components, or a component can include one ormore application programs.

Some embodiments include some, all, or none of the components along withother modules or application components. Still yet, various embodimentsmay incorporate two or more of these components into a single moduleand/or associate a portion of the functionality of one or more of thesecomponents with a different component.

The term “memory” can be any device or mechanism used for storinginformation. In accordance with some embodiments of the presentdisclosure, memory is intended to encompass any type of, but is notlimited to: volatile memory, nonvolatile memory, and dynamic memory. Forexample, memory can be random access memory, memory storage devices,optical memory devices, magnetic media, floppy disks, magnetic tapes,hard drives, SIMMs, SDRAM, DIMMs, RDRAM, DDR RAM, SODIMMS, erasableprogrammable read-only memories (EPROMs), electrically erasableprogrammable read-only memories (EEPROMs), compact disks, DVDs, and/orthe like. In accordance with some embodiments, memory may include one ormore disk drives, flash drives, databases, local cache memories,processor cache memories, relational databases, flat databases, servers,cloud based platforms, and/or the like. In addition, those of ordinaryskill in the art will appreciate many additional devices and techniquesfor storing information can be used as memory.

Memory may be used to store instructions for running one or moreapplications or modules on a processor. For example, memory could beused in some embodiments to house all or some of the instructions neededto execute the functionality of one or more of the modules and/orapplications disclosed in this application.

HTP Microbial Strain Engineering Based Upon Genetic Design Predictions:An Example Workflow

In some embodiments, the present disclosure teaches the directedengineering of new host organisms based on the recommendations of thecomputational analysis systems of the present disclosure.

In some embodiments, the present disclosure is compatible with allgenetic design and cloning methods. That is, in some embodiments, thepresent disclosure teaches the use of traditional cloning techniquessuch as polymerase chain reaction, restriction enzyme digestions,ligation, homologous recombination, RT PCR, and others generally knownin the art and are disclosed in for example: Sambrook et al. (2001)Molecular Cloning: A Laboratory Manual (3^(rd) ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.), incorporated herein by reference.

In some embodiments, the cloned sequences can include possibilities fromany of the HTP genetic design libraries taught herein, for example:promoters from a promoter swap library, SNPs from a SNP swap library,start or stop codons from a start/stop codon exchange library,terminators from a STOP swap library, or sequence optimizations from asequence optimization library.

Further, the exact sequence combinations that should be included in aparticular construct can be informed by the epistatic mapping function.

In other embodiments, the cloned sequences can also include sequencesbased on rational design (hypothesis-driven) and/or sequences based onother sources, such as scientific publications.

In some embodiments, the present disclosure teaches methods of directedengineering, including the steps of i) generating custom-madeSNP-specific DNA, ii) assembling SNP-specific constructs, iii)transforming target host cells with SNP-specific DNA, and iv) loopingout any selection markers (see FIG. 2).

FIG. 6A depicts the general workflow of the strain engineering methodsof the present disclosure, including acquiring and assembling DNA,assembling any necessary vectors, transforming host cells and removingselection markers.

Build Specific DNA Oligonucleotides

In some embodiments, the present disclosure teaches inserting and/orreplacing and/or altering and/or deleting a DNA segment of the host cellorganism. In some aspects, the methods taught herein involve building anoligonucleotide of interest (i.e. a target DNA segment), that will beincorporated into the genome of a host organism. In some embodiments,the target DNA segments of the present disclosure can be obtained viaany method known in the art, including: copying or cutting from a knowntemplate, mutation, or DNA synthesis. In some embodiments, the presentdisclosure is compatible with commercially available gene synthesisproducts for producing target DNA sequences (e.g., GeneArt™, GeneMaker™,GenScript™, Anagen™, Blue Heron™, Entelechon™, GeNOsys, Inc., orQiagen™).

In some embodiments, the target DNA segment is designed to incorporate aSNP into a selected DNA region of the host organism (e.g., adding abeneficial SNP). In other embodiments, the DNA segment is designed toremove a SNP from the DNA of the host organisms (e.g., removing adetrimental or neutral SNP).

In some embodiments, the oligonucleotides used in the inventive methodscan be synthesized using any of the methods of enzymatic or chemicalsynthesis known in the art. The oligonucleotides may be synthesized onsolid supports such as controlled pore glass (CPG), polystyrene beads,or membranes composed of thermoplastic polymers that may contain CPG.Oligonucleotides can also be synthesized on arrays, on a parallelmicroscale using microfluidics (Tian el al., Mol. BioSyst., 5, 714-722(2009)), or known technologies that offer combinations of both (seeJacobsen et al., U.S. Pat. App. No. 2011/0172127).

Synthesis on arrays or through microfluidics offers an advantage overconventional solid support synthesis by reducing costs through lowerreagent use. The scale required for gene synthesis is low, so the scaleof oligonucleotide product synthesized from arrays or throughmicrofluidics is acceptable. However, the synthesized oligonucleotidesare of lesser quality than when using solid support synthesis (See Tianinfra.; see also Staehler et al., U.S. Pat. App. No. 2010/0216648).

A great number of advances have been achieved in the traditionalfour-step phosphoramidite chemistry since it was first described in the1980s (see for example, Sierzchala, et al. J. Am. Chem. Soc., 125,13427-13441 (2003) using peroxy anion deprotection; Hayakawa et al.,U.S. Pat. No. 6,040,439 for alternative protecting groups; Azhayev etal, Tetrahedron 57, 4977-4986 (2001) for universal supports; Kozlov etal., Nucleosides, Nucleotides, and Nucleic Acids, 24 (5-7), 1037-1041(2005) for improved synthesis of longer oligonucleotides through the useof large-pore CPG; and Damha et al., NAR, 18, 3813-3821 (1990) forimproved derivatization).

Regardless of the type of synthesis, the resulting oligonucleotides maythen form the smaller building blocks for longer oligonucleotides. Insome embodiments, smaller oligonucleotides can be joined together usingprotocols known in the art, such as polymerase chain assembly (PCA),ligase chain reaction (LCR), and thermodynamically balanced inside-outsynthesis (TBIO) (see Czar et al. Trends in Biotechnology, 27, 63-71(2009)). In PCA, oligonucleotides spanning the entire length of thedesired longer product are annealed and extended in multiple cycles(typically about 55 cycles) to eventually achieve full-length product.LCR uses ligase enzyme to join two oligonucleotides that are bothannealed to a third oligonucleotide. TBIO synthesis starts at the centerof the desired product and is progressively extended in both directionsby using overlapping oligonucleotides that are homologous to the forwardstrand at the 5′ end of the gene and against the reverse strand at the3′ end of the gene.

Another method of synthesizing a larger double stranded DNA fragment isto combine smaller oligonucleotides through top-strand PCR (TSP). Inthis method, a plurality of oligonucleotides spans the entire length ofa desired product and contain overlapping regions to the adjacentoligonucleotide(s). Amplification can be performed with universalforward and reverse primers, and through multiple cycles ofamplification a full-length double stranded DNA product is formed. Thisproduct can then undergo optional error correction and furtheramplification that results in the desired double stranded DNA fragmentend product.

In one method of TSP, the set of smaller oligonucleotides that will becombined to form the full-length desired product are between 40-200bases long and overlap each other by at least about 15-20 bases. Forpractical purposes, the overlap region should be at a minimum longenough to ensure specific annealing of oligonucleotides and have a highenough melting temperature (Tm) to anneal at the reaction temperatureemployed. The overlap can extend to the point where a givenoligonucleotide is completely overlapped by adjacent oligonucleotides.The amount of overlap does not seem to have any effect on the quality ofthe final product. The first and last oligonucleotide building block inthe assembly should contain binding sites for forward and reverseamplification primers. In one embodiment, the terminal end sequence ofthe first and last oligonucleotide contain the same sequence ofcomplementarity to allow for the use of universal primers.

Assembling DNA Fragments/Cloning Custom Plasmids

In some embodiments, the present disclosure teaches methods forconstructing DNA fragments capable of inserting desired target DNAsections (e.g. containing a particular SNP) into the genome of hostorganisms. FIG. 1 depicts a DNA recombination method of the presentdisclosure for increasing variation in diversity pools. DNA sections,such as genome regions from related species, can be cut via physical orenzymatic/chemical means. The cut DNA regions are melted and allowed toreanneal, such that overlapping genetic regions prime polymeraseextension reactions. Subsequent melting/extension reactions are carriedout until products are reassembled into chimeric DNA, comprisingelements from one or more starting sequences and a promoter. A generalscheme for the entire design, generate, assemble, QC, transform,loop-out and QC process for a SNPswap is shown in FIG. 2. It should benoted that this scheme can be applied to other HTP tools as providedherein (e.g., PROswp, STOPswp). In some embodiments, the presentdisclosure teaches methods of generating linear DNA fragments comprisingthe target DNA, homology arms, and at least one selection marker (seeFIGS. 45 and 46A).

In some embodiments, the present disclosure is compatible with anymethod suited for transformation of DNA fragments into the host organism(e.g., filamentous fungus such as A. niger). In some embodiments, thepresent disclosure teaches use of plasmids or assembly vectors for whicha desired target DNA section can be cloned into and amplified therefrom.When used, the assembly vectors can further comprise any origins ofreplication that may be needed for propagation in a host cell (e.g.,yeast and/or E. coli). In certain instances, the target DNA can beinserted into vectors, constructs or plasmids obtainable from anyrepository or catalogue product, such as a commercial vector (see e.g.,DNA2.0 custom or GATEWAY® vectors). In certain instances, the target DNAcan be inserted into vectors, constructs or plasmids obtainable from anyrepository or catalogue product, such as a commercial vector (see e.g.,DNA2.0 custom or GATEWAY® vectors). The use of plasmids for generatinglinear DNA fragments for ultimately transforming a host cell such as afilamentous fungus host cell can entail synthesizing parts of a targetDNA construct comprising a desired gene to be integrated into a hostgenome, transforming a yeast cell with the parts of the target DNAconstruct along with an assembly vector, isolating the assembledplasmids containing the target DNA construct from said transformed yeastcell, propagating the isolated plasmids in E. coli, and PCR amplifyingthe target DNA construct from E. coli to generate a linear DNA fragmentcomprising a desired gene to be integrated into a host genome prior totransformation of the filamentous fungal host cell.

In an alternative embodiment, assembly or generation of a linear DNAfragment(s) comprising a desired gene to be integrated into a hostgenome (e.g., filamentous fungal cell) can entail using fusion PCR.Fusion PCR can be performed using any fusion PCR method known in the artincluding, for example, the method described in Yu et al, FungalGenetics and Biology, vol 41, pages 973-981 (2004), which is hereinincorporated by reference in its entirety. FIG. 45 depicts a method ofthe use of fusion PCR to generate two linear DNA fragments that comprisea marker gene (i.e., pyrG) split between them. Conceptually, fusion PCRcan be used to generate any of the constructs comprising target genemutations and/or selectable markers genes provided herein.

The linear DNA fragments for use in the methods provided herein cancomprise markers for selection and/or counter-selection as describedherein. The markers can be any markers known in the art and/or providedherein. The linear DNA fragments can further comprise any regulatorysequence(s) provided herein. The regulatory sequence can be anyregulatory sequence known in the art or provided herein such as, forexample, a promoter, start, stop, signal, secretion and/or terminationsequence used by the genetic machinery of the host cell (e.g.,filamentous fungal cell).

In some embodiments, the assembly/cloning methods of the presentdisclosure may employ at least one of the following assembly strategies:i) type II conventional cloning, ii) type II S-mediated or “Golden Gate”cloning (see, e.g., Engler, C., R. Kandzia, and S. Marillonnet. 2008 “Aone pot, one step, precision cloning method with high-throughputcapability”. PLos One 3:e3647; Kotera, I., and T. Nagai. 2008 “Ahigh-throughput and single-tube recombination of crude PCR productsusing a DNA polymerase inhibitor and type IIS restriction enzyme.” JBiotechnol 137:1-7.; Weber, E., R. Gruetzner, S. Werner, C. Engler, andS. Marillonnet. 2011 Assembly of Designer TAL Effectors by Golden GateCloning. PloS One 6:e19722), iii) GATEWAY® recombination, iv) TOPO®cloning, exonuclease-mediated assembly (Aslanidis and de Jong 1990.“Ligation-independent cloning of PCR products (LIC-PCR).” Nucleic AcidsResearch, Vol. 18, No. 20 6069), v) homologous recombination, vi)non-homologous end joining, vii) Gibson assembly (Gibson et al., 2009“Enzymatic assembly of DNA molecules up to several hundred kilobases”Nature Methods 6, 343-345) or a combination thereof. Modular type IISbased assembly strategies are disclosed in PCT Publication WO2011/154147, the disclosure of which is incorporated herein byreference.

In some embodiments, the present disclosure teaches cloning vectors withat least one selection marker. Various selection marker genes are knownin the art often encoding antibiotic resistance function for selectionin prokaryotic (e.g., against ampicillin, kanamycin, tetracycline,chloramphenicol, zeocin, spectinomycin/streptomycin) or eukaryotic cells(e.g. geneticin, neomycin, hygromycin, puromycin, blasticidin, zeocin)under selective pressure. Other marker systems allow for screening andidentification of wanted or unwanted cells such as the well-knownblue/white screening system used in bacteria to select positive clonesin the presence of X-gal or fluorescent reporters such as green or redfluorescent proteins expressed in successfully transduced host cells.Another class of selection markers most of which are only functional inprokaryotic systems relates to counter selectable marker genes oftenalso referred to as “death genes” which express toxic gene products thatkill producer cells. Examples of such genes include sacB, rpsL(strA),tetAR, pheS, thyA, gata-1, or ccdB, the function of which is describedin (Reyrat et al. 1998 “Counterselectable Markers: Untapped Tools forBacterial Genetics and Pathogenesis.” Infect Immun. 66(9): 4011-4017).

FIG. 18 depicts a workflow associated with DNA assembly according to oneembodiment of the present disclosure. This process can be divided upinto 4 stages: parts generation, plasmid/construct assembly,plasmid/construct QC, and plasmid/construct preparation fortransformation. During parts generation, oligos designed by LaboratoryInformation Management System (LIMS) are ordered from an oligosequencing vendor and used to amplify the target sequences from the hostorganism via PCR. These PCR parts are cleaned to remove contaminants andassessed for success by fragment analysis, in silico quality controlcomparison of observed to theoretical fragment sizes, and DNAquantification. As shown in FIG. 18, in one embodiment, the parts aretransformed into yeast along with an assembly vector and assembled intoplasmids via homologous recombination. Assembled plasmids are isolatedfrom yeast and transformed into a separate yeast host cell forsubsequent assembly quality control and amplification. During plasmidassembly quality control, several replicates of each plasmid areisolated, amplified using Rolling Circle Amplification (RCA), andassessed for correct assembly by enzymatic digest and fragment analysis.Correctly assembled plasmids identified during the QC process are hitpicked to generate permanent stocks and the specific gene constructincluding any flanking sequences necessary to facilitate genomeintegration are then PCR amplified from the plasmid to generate linearDNA fragments that are quantified and QC'd via fragment analysis priorto transformation into the target host organism (e.g., filamentousfungal host cell). As also shown in FIG. 18, in a separate embodiment,the parts are subjected to fusion PCR (see FIGS. 45 and 46A-B forexample) to generate linear DNA fragments, which are QC'd via fragmentand sequence analysis prior to transformation into the target hostorganism (e.g., filamentous fungal host cell).

Protoplasting Methods

In one embodiment, the methods and systems provided herein require thegeneration of protoplasts from coenocytic organisms (e.g., filamentousfungal cells) as provided herein. Suitable procedures for preparation ofprotoplasts can be any known in the art including, for example, thosedescribed in EP 238,023 and Yelton et al. (1984, Proc. Natl. Acad. Sci.USA 81:1470-1474). In one embodiment, protoplasts are generated bytreating a pre-cultivated culture of filamentous fungal cells with oneor more lytic enzymes or a mixture thereof. The lytic enzymes can be abeta-glucanase and/or a polygalacturonase. In one embodiment, the enzymemixture for generating protoplasts is VinoTaste concentrate. Many of theparameters utilized to pre-cultivate cultures of coenocytic organisms(e.g., filamentous fungal cells) and subsequently generate and utilizeprotoplasts therefrom for use in the methods and compositions providedherein can be varied. For example, there can be variations of inoculumsize, inoculum method, pre-cultivation media, pre-cultivation times,pre-cultivation temperatures, mixing conditions, washing buffercomposition, dilution ratios, buffer composition during lytic enzymetreatment, the type and/or concentration of lytic enzyme used, the timeof incubation with lytic enzyme, the protoplast washing proceduresand/or buffers, the concentration of protoplasts and/or polynucleotideand/or transformation reagents during the actual transformation, thephysical parameters during the transformation, the procedures followingthe transformation up to the obtained transformants. In some cases,these variations can be utilized to optimize the number of protoplastsand the transformation efficiency. In one embodiment, the coenocyticorganism is a filamentous fungal cell as provided herein (e.g., A.niger). Further to this embodiment, the pre-cultivation media can be YPDor complete media. The volume of pre-cultivation media can be at least,at most or about 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml,400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 650 ml, 700 ml, 750 ml, 800 ml,850 ml, 900 ml, 950 ml or 1000 ml. The volume of pre-cultivation mediacan be from about 50 ml to about 100 ml, about 100 ml to about 150 ml,about 150 ml to about 200 ml, about 200 ml to about 250 ml, about 250 mlto about 300 ml, about 300 ml to about 350 ml, about 350 ml to about 400ml, about 400 ml to about 450 ml, about 450 ml to about 500 ml, about500 ml to about 550 ml, about 550 ml to about 600 ml, about 600 ml toabout 650 ml, about 650 ml to about 700 ml, about 700 ml to about 750ml, about 750 ml to about 800 ml, about 800 ml to about 850 ml, about850 ml to about 900 ml, about 900 ml to about 950 ml or about 950 ml toabout 1000 ml. In some cases, a plurality of cultures are cultivated andsubsequently subjected to protoplasting. The plurality of cultures canbe 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 50, 75, 100, 150,200, 300, 400, 500 or more. In one embodiment, a pre-cultivationpreparation is prepared by inoculating 100 ml of rich media (e.g., YPDor complete media) with 10⁶ spores/ml and incubating the pre-cultivationpreparation between 14-18 hours at 30° C. In another embodiment, apre-cultivation preparation is prepared by inoculating 500 ml of richmedia (e.g., Yeast Mold Broth, YPD or complete media) with at least 10⁶spores/ml and incubating the pre-cultivation preparation between 14-18hours at 30° C. Prior to protoplasting, the coenocytic organism can beisolated by any method known in the art such as, for examplecentrifugation. In one embodiment, the coenocytic organism isfilamentous fungus (e.g., A. niger). Further to this embodiment, YeastMold Broth (YMB) is inoculated with 10⁶ spores/ml of the filamentousfungal cells and grown for 16 hours at 30° C. Further still to thisembodiment, the filamentous fungal cells grown in the precultivationpreparation can be isolated by centrifugation. The pre-cultivationpreparations provided herein for use in the methods and compositionsprovided herein can produce an amount of hyphae for subsequentprotoplasting of about, at least or more than 0.5 g, 1 g, 1.5 g, 2 g,2.5 g, 3 g, 3.5 g, 4 g or 5 g of wet weight. Pre-cultivation/cultivationof the coenocytic organism (e.g., filamentous fungus) can be part of aworkflow in a high-throughput system (HTP) such as depicted in FIG. 28.The HTP system can be automated or semi-automated. As shown in FIG. 28,pre-cultivation of the organism can entail inoculating a small scalevolume (e.g., 100 ml) of sporulation media (PDAmedia in FIG. 28) with10⁶ spores/ml of the organism (e.g., A. niger) and growing for 14-16hours at 30° C. As shown in FIG. 28, during pre-cultivation, theworkflow can contain a step whereby an enzyme solution for generatingprotoplasts from the pre-cultivated organism (e.g., A. niger) isgenerated. The enzyme solution can consist of Vinotaste pro (Novozymes)enzyme mix in phosphate buffer comprising 1.2 M MgSO₄. Followingpre-cultivation, hyphae can be collected following filtration through aMiracloth and a large-scale culture can be cultivated by inoculatingabout 500 ml of complete media in a 2.8 L flask with 10 ul to 20 ml ofthe collected hyphae. Inoculum size can be variable based on the OD ofthe culture obtained from the pre-cultivation step. The large scaleculture can be grown for 6-18 hours at either 30° C. or 18° C. at 80%humidity with shaking at 200 rpms. Following cultivation, the culture(s)can be isolated by centrifugation following by one or more washes andresuspended. In one embodiment, the cultures are resuspended in aprotoplasting buffer as described herein and subjected to protoplastingas described herein. Centrifugation can be performed in 500 mlcentrifuge tubes at 4° C. for 10-15 minutes at 5500-6100×g. Each of theone or more washes can be performed in 10-50 ml of wash buffer (e.g.,water with 10% glycerol) followed by centrifugation at 4° C. for 10-15minutes at 5500-6100×g.

Following isolation as described above, the coenocytic organism (e.g.,filamentous fungal cells such as A. niger) can be resuspended inprotoplasting buffer such that the protoplasting buffer comprises one orenzymes as provided herein (e.g., VinoTaste pro concentrate (Novozymes))for generating protoplasts. In one embodiment, the protoplasting bufferhas a high concentration of osmolite (e.g., greater than or equal to 1 Mof an osmolite such as MgSO₄). In embodiments utilizing a protoplastingbuffer with a high osmolite concentration (e.g., 1.2 M MgSO₄), theincubation time for the enzymatic treatment (e.g., VinoTaste proconcentrate (Novozymes)) can be from about 14-16 hours at about 30° C.The volume of protoplasting buffer used for resuspension can be 50 ml,100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml,550 ml, 600 ml, 650 ml, 700 ml, 750 ml, 800 ml, 850 ml, 900 ml, 950 mlor 1000 ml. The volume of protoplasting buffer used for resuspension canbe can be from about 50 ml to about 100 ml, about 100 ml to about 150ml, about 150 ml to about 200 ml, about 200 ml to about 250 ml, about250 ml to about 300 ml, about 300 ml to about 350 ml, about 350 ml toabout 400 ml, about 400 ml to about 450 ml, about 450 ml to about 500ml, about 500 ml to about 550 ml, about 550 ml to about 600 ml, about600 ml to about 650 ml, about 650 ml to about 700 ml, about 700 ml toabout 750 ml, about 750 ml to about 800 ml, about 800 ml to about 850ml, about 850 ml to about 900 ml, about 900 ml to about 950 ml or about950 ml to about 1000 ml. In one embodiment, filamentous fungal cells aregrown in 500 ml of rich media (e.g., YPD or complete media) as shown,for example, in FIG. 28, and hyphae (can be about 1 g wet mass as shownin FIG. 28) are isolated by filtration through a Miracloth, rinsing with100 ml of wash buffer (e.g., 100 mM sodium phosphate buffer with 1.2 MMgSO₄, pH 5.5) and resuspended in about 500 ml of protoplasting buffer(e.g., 100 mM sodium phosphate buffer with 1.2 M MgSO₄ pH 5.5 in FIG.28) comprising a protoplasting enzyme mixture (e.g., VinoTaste proconcentrate (Novozymes)) in a 1 L bottle. The hyphae in the enzymesolution can be incubated for 14-16 hours at 30° C. with shaking at 140rpm with continued monitoring of protoplast formation via microscopicexamination.

In one embodiment, one or more chemical inhibitors of the NHEJ pathwayare added to a protoplasting buffer as provided. The one or morechemical inhibitors can be selected from W-7, chlorpromazine, vanillin,Nu7026, Nu7441, mirin, SCR7, AG14361 or any combination thereof.Addition of the one or more chemical inhibitors to the protoplastingbuffer can occur at any point during the protoplasting procedure. In oneembodiment, treatment with the one or more chemical inhibitors is forthe entire protoplasting procedure. In a separate embodiment, treatmentwith the one or more chemical inhibitors is for less than the entireprotoplasting procedure. Treatment with the one or more chemicalinhibitors can be for about 1, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150,180, 210, 240, 270 or 300 minutes. In one embodiment, the co-enocyticcells (e.g., filamentous fungal cells) are treated with W-7. In anotherembodiment, the co-enocytic cells (e.g., filamentous fungal cells) aretreated with SCR-7.

Following enzymatic treatment, the protoplasts can be isolated usingmethods known in the art. Prior to isolation of protoplasts, undigestedhyphal fragments can be removed by filtering the mixture through aporous barrier (such as Miracloth) in which the pores range in size from20-100 microns in order to produce a filtrate of filtered protoplasts.In one embodiment, the filtered protoplasts are then centrifuged atmoderate levels of centripetal force to cause the protoplasts to pelletto the bottom of the centrifuge tube. The centripetal force can be fromabout 500-1500×g. In a preferred embodiment, the centripetal force usedis generally below 1000×g (e.g., 800×g for 5 minutes as shown in FIG.28). In a separate embodiment, a buffer of substantially lower osmoticstrength is gently applied to the surface of the protoplasts (e.g.,filtered protoplasts) following generation of protoplasts in aprotoplasting buffer comprising a high concentration of osmolite.Examples of buffers of substantially lower osmotic strength includebuffers (e.g., Tris buffer) comprising 1M Sorbitol, 1M NaCl, 0.6MAmmonium Sulfate or 1M KCl. In one embodiment, as shown in FIG. 28, thelower osmotic strength buffer for use in the methods provided herein isa Sorbitol-Tris (ST) buffer that comprises 0.4 M sorbitol and has a pHof 8. This layered preparation can then be centrifuged, which can causethe protoplasts to accumulate at a layer in the tube in which they areneutrally buoyant. Protoplasts can then be isolated from this layer forfurther processing (e.g., storage and/or transformation). In yet anotherembodiment, the protoplasts (e.g., filtered protoplasts) generated in aprotoplasting buffer comprising a high concentration of osmolite (e.g.,100 mM phosphate buffer comprising 1.2M MgSO₄, pH 5.5) are transferredto an elongated collection vessel (e.g., graduated cylinder) and abuffer of lower osmolarity as provided herein (e.g., 0.4M ST buffer, pH8) is overlaid on the surface of the protoplasts (e.g., filteredprotoplasts) to generate a layer at which the protoplasts are neutrallybuoyant. The combination of the buffers of differing osmolarity in theelongated collection vessel (e.g., graduated cylinder) can facilitatethe protoplasts ‘floating’ to the surface of the elongated collectionvessel (e.g., graduated cylinder; FIG. 27). Once at the top of thecollection vessel, the protoplasts can be isolated. In one embodiment, a500 ml pre-cultivation preparation of coenocytic organisms (e.g.,filamentous fungal cells such as A. niger) grown and subjected toprotoplasting as provided herein yields about 25 ml of protoplasts.

Following protoplast isolation, the remaining enzyme containing buffercan be removed by resuspending the protoplasts in an osmotic buffer(e.g., 1M sorbitol buffered using 10 mM TRIS, pH 8) and recollected bycentrifugation as shown in FIG. 28. This step can be repeated. Aftersufficient removal of the enzyme containing buffer, the protoplasts canbe further washed in osmotically stabilized buffer also containingCalcium chloride (e.g., 1M sorbitol buffered using 10 mM TRIS, pH 8, 50mM CaCl₂) one or more times (see, for example, FIG. 28).

Following isolation and washing, the protoplasts can be resuspended inan osmotic stabilizing buffer. The composition of such buffers can varydepending on the species, application and needs. However, typicallythese buffers contain either an organic component like sucrose, citrate,mannitol or sorbitol between 0.5 and 2 M. More preferably between 0.75and 1.5 M; most preferred is 1 M. Otherwise these buffers contain aninorganic osmotic stabilizing component like KCl, (NH₄)₂SO₄. MgSO₄, NaClor MgCl₂ in concentrations between 0.1 and 1.5 M. Preferably between 0.2and 0.8 M; more preferably between 0.3 and 0.6 M, most preferably 0.4 M.The most preferred stabilizing buffers are STC (sorbitol, 0.8 M;CaCl.sub.2, 25 mM; Tris, 25 mM; pH 8.0) or KCl-citrate (KCl, 0.3-0.6 M;citrate, 0.2% (w/v)). The protoplasts can be used in a concentrationbetween 1×10⁵ and 1×10¹⁰ cells/ml or between 1-3×10⁷ protoplasts per ml.Preferably, the concentration is between 1×10⁶ and 1×10⁹; morepreferably the concentration is between 1×10⁷ and 5×10⁸; most preferablythe concentration is 1×10⁸ cells/ml. To increase the efficiency oftransfection, carrier DNA (as salmon sperm DNA or non-coding vector DNA)may be added to the transformation mixture. DNA is used in aconcentration between 0.01 and 10 ug; preferably between 0.1 and 5 ug,even more preferably between 0.25 and 2 ug; most preferably between 0.5and 1 ug.

In one embodiment, following generation and subsequent isolation andwashing, the protoplasts are mixed with one or more cryoprotectants. Thecryoprotectants can be glycols, dimethyl sulfoxide (DMSO), polyols,sugars, 2-Methyl-2,4-pentanediol (MPD), polyvinylpyrrolidone (PVP),methylcellulose, C-linked antifreeze glycoproteins (C-AFGP) orcombinations thereof. Glycols for use as cryoprotectants in the methodsand systems provided herein can be selected from ethylene glycol,propylene glycol, polypropylene glycol (PEG), glycerol, or combinationsthereof. Polyols for use as cryoprotectants in the methods and systemsprovided herein can be selected from propane-1,2-diol, propane-1,3-diol,1,1,1-tris-(hydroxymethyl)ethane (THME), and2-ethyl-2-(hydroxymethyl)-propane-1,3-diol (EHMP), or combinationsthereof. Sugars for use as cryoprotectants in the methods and systemsprovided herein can be selected from trehalose, sucrose, glucose,raffinose, dextrose or combinations thereof. In one embodiment, theprotoplasts are mixed with DMSO. DMSO can be mixed with the protoplastsat a final concentration of at least, at most, less than, greater than,equal to, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% w/v orv/v. The protoplasts/cryoprotectant (e.g., DMSO) mixture can bedistributed to microtiter plates prior to storage. Theprotoplast/cryoprotectant (e.g., DMSO) mixture can be stored at anytemperature provided herein for long-term storage (e.g., several hours,day(s), week(s), month(s), year(s)) as provided herein such as, forexample −20° C. or −80° C. In one embodiment, an additionalcryoprotectant (e.g., PEG) is added to the protoplasts/DMSO mixture. Inyet another embodiment, the additional cryoprotectant (e.g., PEG) isadded to the protoplast/DMSO mixture prior to storage. The PEG can beany PEG provided herein and can be added at any concentration (e.g., w/vor v/v) as provided herein. In one embodiment, the PEG solution isprepared as 40% w/v in STC buffer. 20% v/v of this 40% PEG-STC can thenbe added to the protoplasts. For example, 800 microliters of 1.25×10⁷protoplasts would have 200 microliters of 40% PEG-STC giving a finalvolume of 1 ml. Seventy microliters of DMSO can then be added to this 1ml to bring this prep to 7% v/v DMSO.

Any pre-cultivation, cultivation and/or protoplasting protocol providedherein can be performed in a high-throughput manner. For example,pre-cultivation, cultivation and protoplasting can be performed as partof a workflow such that said workflow represents a portion of ahigh-throughput (HTP) protocol such as depicted in FIG. 28. Thehigh-throughput protocol can utilized automated liquid handling for anyand/or all steps.

Transformation of Host Cells

In some embodiments, the vectors or constructs of the present disclosuremay be introduced into the host cells (e.g., filamentous fungal cells orprotoplasts derived therefrom) using any of a variety of techniques,including transformation, transfection, transduction, viral infection,gene guns, or Ti-mediated gene transfer (see Christie, P. J., andGordon, J. E., 2014 “The Agrobacterium Ti Plasmids” Microbiol SPectr.2014; 2(6); 10.1128). Particular methods include calcium phosphatetransfection, DEAE-Dextran mediated transfection, lipofection, orelectroporation (Davis, L., Dibner, M., Battey, I., 1986 “Basic Methodsin Molecular Biology”). Other methods of transformation include, forexample, lithium acetate transformation and electroporation see, e.g.,Gietz et al., Nucleic Acids Res. 27:69-74 (1992); Ito et al., J.Bacterol. 153:163-168 (1983); and Becker and Guarente, Methods inEnzymology 194:182-187 (1991). In some embodiments, transformed hostcells are referred to as recombinant host strains.

In some embodiments, the present disclosure teaches high-throughputtransformation of cells using the 96-well plate robotics platform andliquid handling machines of the present disclosure.

In one embodiment, the methods and systems provided herein require thetransfer of nucleic acids to protoplasts derived from filamentous fungalcells as described herein. In another embodiment, the transformationutilized by the methods and systems provided herein is high-throughputin nature and/or is partially or fully automated as described herein.The partially or fully automated method can entail the use of automatedliquid handling one or more liquid handling steps as provided herein.Further to this embodiment, the transformation is performed by addingconstructs or expression constructs as described herein to the wells ofa microtiter plate followed by aliquoting protoplasts generated by themethods provided herein to each well of the microtiter plate. Suitableprocedures for transformation/transfection of protoplasts can be anyknown in the art including, for example, those described ininternational patent applications PCT/NL99/00618, PCT/EP99/202516,Finkelstein and Ball (eds.), Biotechnology of filamentous fungi,technology and products, Butterworth-Heinemann (1992), Bennett andLasure (eds.) More Gene Manipulations in fungi, Academic Press (1991),Turner, in: Puhler (ed), Biotechnology, second completely revisededition, VHC (1992) protoplast fusion, and the Ca-PEG mediatedprotoplast transformation as described in EP635574B. Alternatively,transformation of the filamentous fungal host cells or protoplastsderived therefrom can also be performed by electroporation such as, forexample, the electroporation described by Chakraborty and Kapoor,Nucleic Acids Res. 18:6737 (1990), Agrobacterium tumefaciens-mediatedtransformation, biolistic introduction of DNA such as, for example, asdescribed in Christiansen et al., Curr. Genet. 29:100 102 (1995); Durandet al., Curr. Genet. 31:158 161 (1997); and Barcellos et al., Can. J.Microbiol. 44:1137 1141 (1998) or “magneto-biolistic” transfection ofcells such as, for example, described in U.S. Pat. Nos. 5,516,670 and5,753,477. In one embodiment, the transformation procedure used in themethods and systems provided herein is one amendable to beinghigh-throughput and/or automated as provided herein such as, forexample, PEG mediated transformation.

Transformation of the protoplasts generated using the methods describedherein can be facilitated through the use of any transformation reagentknown in the art. Suitable transformation reagents can be selected fromPolyethylene Glycol (PEG), FUGENE® HD (from Roche), Lipofectamine® orOLIGOFECTAMINE® (from Invitrogen), TRANSPASS® D1 (from New EnglandBiolabs), LYPOVEC® or LIPOGEN® (from Invivogen). In one embodiment, PEGis the most preferred transformation/transfection reagent. PEG isavailable at different molecular weights and can be used at differentconcentrations. Preferably, PEG 4000 is used between 10% and 60%, morepreferably between 20% and 50%, most preferably at 40%. In oneembodiment, the PEG is added to the protoplasts prior to storage asdescribed herein.

Looping Out of Selected Sequences

In some embodiments, the present disclosure teaches methods of loopingout selected regions of DNA from the host organisms. The looping outmethod can be as described in Nakashima et al. 2014 “Bacterial CellularEngineering by Genome Editing and Gene Silencing.” Int. J. Mol. Sci.15(2), 2773-2793. In some embodiments, the present disclosure teacheslooping out selection markers from positive transformants. Looping outdeletion techniques are known in the art, and are described in (Tear etal. 2014 “Excision of Unstable Artificial Gene-Specific inverted RepeatsMediates Scar-Free Gene Deletions in Escherichia coli.” Appl. Biochem.Biotech. 175:1858-1867). The looping out methods used in the methodsprovided herein can be performed using single-crossover homologousrecombination or double-crossover homologous recombination. In oneembodiment, looping out of selected regions as described herein canentail using single-crossover homologous recombination as describedherein.

First, loop out constructs are inserted into selected target regionswithin the genome of the host organism (e.g., via homologousrecombination, CRISPR, or other gene editing technique). In oneembodiment, double-crossover homologous recombination is used between aconstruct or constructs and the host cell genome in order to integratethe construct or constructs such as depicted in FIG. 3. The insertedconstruct or constructs can be designed with a sequence which is adirect repeat of an existing or introduced nearby host sequence, suchthat the direct repeats flank the region of DNA slated for looping-outand deletion. Once inserted, cells containing the loop out construct orconstructs can be counter selected for deletion of the selection region(e.g., see FIG. 4; lack of resistance to the selection gene). Furtherillustrations of the loop-in and loop-out process are depicted in FIGS.36-37.

Persons having skill in the art will recognize that the description ofthe loopout procedure represents but one illustrative method fordeleting unwanted regions from a genome. Indeed the methods of thepresent disclosure are compatible with any method for genome deletions,including but not limited to gene editing via CRISPR, TALENS, FOK, orother endonucleases. Persons skilled in the art will also recognize theability to replace unwanted regions of the genome via homologousrecombination techniques

Constructs for Transformation

In one embodiment, the methods and systems provided herein entail thetransformation or transfection of filamentous fungal cells orprotoplasts derived therefrom with at least one nucleic acid. Thetransformation or transfection can be using of the methods and reagentsdescribed herein. The generation of the protoplasts can be performedusing any of the methods provided herein. The protoplast generationand/or transformation can be high-throughput and/or automated asprovided herein. The nucleic acid can be DNA, RNA or cDNA. The nucleicacid can be a polynucleotide. The nucleic acid or polynucleotide for usein transforming a filamentous fungal cell or protoplast derivedtherefrom using the methods and systems provided herein can be anendogenous gene or a heterologous gene relative to the variant strainand/or the parental strain. The endogenous gene or heterologous gene canencode a product or protein of interest as described herein. Asdescribed herein, the protein of interest can refer to a polypeptidethat is desired to be expressed in a filamentous fungus. Such a proteincan be an enzyme, a substrate-binding protein, a surface-active protein,a structural protein, or the like, and can be expressed at high levels,and can be for the purpose of commercialization. The protein of interestcan be expressed intracellularly or as a secreted protein. Theendogenous gene or heterologous gene can comprise a mutation and/or beunder the control of or operably linked to one or more genetic controlor regulatory elements. The mutation can be any mutation provided hereinsuch as, for example, an insertion, deletion, substitution and/or singlenucleotide polymorphism. The one or more genetic control or regulatoryelements can be a promoter sequence and/or a terminator sequence. Theendogenous gene or heterologous gene can be present on one expressionconstruct or split across multiple expression constructs such as shownin FIGS. 36-38. When split across multiple expression constructs, eachportion of the endogenous gene or heterologous gene can comprise amutation and/or be under the control of or operably linked to one ormore genetic control or regulatory elements. In one embodiment, anendogenous gene or heterologous gene is bipartite, wherein saidendogenous gene or heterologous gene is split into two portions suchthat each of said two portions is present on a separate construct. Inone embodiment, the gene is FungiSNP_9 (SEQ ID NO: 11), FungiSNP_12 (SEQID NO: 12), FungiSNP_18 (SEQ ID NO: 13) or FungiSNP_40 (SEQ ID NO: 14).In another embodiment, the gene is FungiSNP_9 (SEQ ID NO: 11),FungiSNP_12 (SEQ ID NO: 12), FungiSNP_18 (SEQ ID NO: 13) or FungiSNP_40(SEQ ID NO: 14) fused to or operably linked to any of the promoters fromTable 1. In one embodiment, the gene is FungiSNP_18 (SEQ ID NO: 13). Inanother embodiment, the gene is FungiSNP_18 (SEQ ID NO: 13) fused to oroperably linked to the man8p or amy8p promoter from Table 1.

The promoter sequence and/or terminator sequence can be endogenous orheterologous relative to the variant strain and/or the parental strain.Promoter sequences can be operably linked to the 5′ termini of thesequences to be expressed. A variety of known fungal promoters arelikely to be functional in the host strains of the disclosure such as,for example, the promoter sequences of C1 endoglucanases, the 55 kDacellobiohydrolase (CBH1), glyceraldehyde-3-phosphate dehydrogenase A, C.lucknowense GARG 27K and the 30 kDa xylanase (Xy1F) promoters fromChrysosporium, as well as the Aspergillus promoters described in, e.g.U.S. Pat. Nos. 4,935,349; 5,198,345; 5,252,726; 5,705,358; and5,965,384; and PCT application WO 93/07277. In one embodiment, thepromoters for use in the methods and systems provided herein areinducible promoters. The inducible promoters can be any promoter whosetranscriptional activity is regulated by the presence or absence of achemical such as for example, alcohol, tetracycline, steroids, metal orother compounds known in the art. The inducible promoters can be anypromoter whose transcriptional activity is regulated by the presence orabsence of light or low or high temperatures. In one embodiment, theinducible promoter is catabolite repressed by glucose (see FIG. 37 forexample) such as, for example, the promoter for the A. niger amylase Bgene. In one embodiment, the inducible promoters are selected fromfilamentous fungal genes such as the srpB gene, the amyB gene, the manBgene or the mbfA gene. In one embodiment, the inducible promoter isselected form the promoters listed in Table 1.

Terminator sequences can be operably linked to the 3′ termini of thesequences to be expressed. A variety of known fungal terminators arelikely to be functional in the host strains of the disclosure. Examplesare the A. nidulans trpC terminator, A. niger alpha-glucosidaseterminator, A. niger glucoamylase terminator, Mucor miehei carboxylprotease terminator (see U.S. Pat. No. 5,578,463), Chrysosporiumterminator sequences, e.g. the EG6 terminator, and the Trichodermareesei cellobiohydrolase terminator. In one embodiment, the terminatorsequences are direct repeats (DRs). In one embodiment, a transcriptionalterminator sequence of the present disclosure can be selected from aterminator sequence listed in Table 1.1 or an orthologue of atermination sequence provided in Table 1.1. For example, if the hostcell is an Aspergillus, the termination sequence can be an orthologue ofa non-Aspergillus termination sequence selected from Table 1.1.

In one embodiment, a protoplast generated from a filamentous fungal cellis co-transformed with two or more nucleic acids or polynucleotides.Further to this embodiment, at least one of the two or morepolynucleotides is an endogenous gene or a heterologous gene relative tothe filamentous fungal strain from which the protoplast was generatedand at least one of the two or more polynucleotides is a gene for aselectable marker. The selectable marker gene can be any selectablemarker as provided herein. As described herein, each of the two or morenucleic acids or polynucleotides can be split into separate portionssuch that each separate portion is present on a separate construct (seeFIGS. 36-38).

In one embodiment, each nucleic acid or polynucleotide for use intransforming or transfecting a filamentous fungal cell or protoplastderived therefrom comprises sequence homologous to DNA sequence presentin a pre-determined target locus of the genome of the filamentous fungalcell or protoplast derived therefrom that is to be transformed on eithera 5′, a 3′ or both a 5′ and a 3′ end of the nucleic acid orpolynucleotide. The nucleic acid or polynucleotide can be an endogenousgene or heterologous gene relative to the filamentous fungal cell usedfor transformation or a selectable marker gene such that sequencehomologous to a pre-determined locus in the filamentous fungal host cellgenome flanks the endogenous, heterologous, or selectable marker gene.In one embodiment, each nucleic acid or polynucleotide is cloned into acloning vector using any method known in the art such as, for example,pBLUESCRIPT® (Stratagene). Suitable cloning vectors can be the ones thatare able to integrate at the pre-determined target locus in thechromosomes of the filamentous fungal host cell used. Preferredintegrative cloning vectors can comprise a DNA fragment, which ishomologous to the DNA sequence to be deleted or replaced for targetingthe integration of the cloning vector to this pre-determined locus. Inorder to promote targeted integration, the cloning vector can belinearized prior to transformation of the host cell or protoplastsderived therefrom. Preferably, linearization is performed such that atleast one but preferably either end of the cloning vector is flanked bysequences homologous to the DNA sequence to be deleted or replaced. Insome cases, short homologous stretches of DNA may be added for examplevia PCR on both sides of the nucleic acid or polynucleotide to beintegrated. The length of the homologous sequences flanking the nucleicacid or polynucleotide sequence to be integrated is preferably less than2 kb, even preferably less, than 1 kb, even more preferably less than0.5 kb, even more preferably less than 0.2 kb, even more preferably lessthan 0.1 kb, even more preferably less than 50 bp and most preferablyless than 30 bp. The length of the homologous sequences flanking thenucleic acid or polynucleotide sequence to be integrated can vary fromabout 30 bp to about 1000 bp, from about 30 bp to about 700 bp, fromabout 30 bp to about 500 bp, from about 30 bp to about 300 bp, fromabout 30 bp to about 200 bp, and from about 30 bp to about 100 bp. Thenucleic acids or polynucleotides for use in transforming filamentousfungal cells or protoplasts derived therefrom can be present asexpression cassettes. In one embodiment, the cloning vector is pUC19.Further to this embodiment, a cloning vector containing a markersequence as provided herein can be associated with targeting sequence bybuilding the construct through using a Gibson assembly as known in theart. Alternatively, the targeting sequence can be added by fusion PCR.Targeting sequence for co-transformation that is not linked to a markermay be amplified from genomic DNA.

In theory, all loci in the filamentous fungi genome could be chosen fortargeted integration of the expression cassettes comprising nucleicacids or polynucleotides provided herein. Preferably, the locus whereintargeting will take place is such that when the wild type gene presentat this locus has been replaced by the gene comprised in the expressioncassette, the obtained mutant will display a change detectable by agiven assay such as, for example a selection/counterselection scheme asdescribed herein. In one embodiment, the protoplasts generated fromfilamentous fungal cells as described herein are co-transformed with afirst construct or expression cassette and a second construct orexpression cassette such that the first construct or expression cassetteis designed to integrate into a first locus of the protoplast genome,while the second construct or expression cassette is designed tointegrate into a second locus of the protoplast genome. To facilitateintegration into the first locus and second locus, the first constructor expression cassette is flanked by sequence homologous to the firstlocus, while the second construct or expression cassette is flanked bysequence homologous to the second locus. In one embodiment, the firstconstruct or expression cassette comprises sequence for an endogenousgene, while the second construct comprises sequence for a selectablemarker gene. Further to this embodiment, the second locus containssequence for an additional selectable marker gene present in theprotoplast genome used in the methods and systems provided herein, whilethe first locus contains sequence for the endogenous target gene presentin the protoplast genome used in the methods and systems providedherein. In a separate embodiment, the first construct or expressioncassette comprises sequence for an endogenous gene or a heterologousgene, while the second construct comprises sequence for a firstselectable marker gene. Further to this separate embodiment, the secondlocus contains sequence for a second selectable marker gene that ispresent in the protoplast genome used in the methods and systemsprovided herein, while the first locus contains sequence for a thirdselectable marker gene that is present in the protoplast genome used inthe methods and systems provided herein. In each of the aboveembodiments, the endogenous gene and/or heterologous gene can comprise amutation (e.g., SNP) and/or a genetic control or regulatory element asprovided herein.

Purification of Homokaryotic Protoplasts

As will be appreciated by those skilled in the art, protoplasts derivedfrom filamentous fungal can often contain more than one nucleus suchthat subsequent transformation with a construct (e.g., insert DNAfragment) as provided herein can produce protoplasts that areheterokaryotic such that the construct (e.g., insert DNA fragment) isincorporated into only a subset of the multiple nuclei present in theprotoplast. In order to reduce the number or percentage ofheterokaryotic protoplasts following transformation, strategies can beemployed to increase the percentage of mononuclear protoplasts in apopulation of protoplasts derived from filamentous fungal host cellsprior to transformation such as, for example, using the method describedin Roncero et al., 1984, Mutat. Res. 125:195, the contents of which areherein incorporated by reference in its entirety.

In another embodiment, provided herein is a method for isolating clonalpopulations derived from individual spores. In some cases, theindividual fungal spores are sporulated from protoplasts derived fromfungal strains following genetic perturbation of said protoplasts. Themethods for isolating the clonal populations derived from the individualspores can facilitates or aid in the isolation of homokaryotic fungalstrains following genetic perturbation using any of the methods providedherein. Further to this embodiment, a plurality of spores (e.g., sporesultimately derived from filamentous fungal cells or strains) can beresuspended to generate a liquid suspension and individual spores indiscrete volumes of the liquid suspension can be placed or distributedinto the wells or reaction areas of a substrate such as, for example, amicrotiter plate. The microtiter plate can be a 96 well, 384 well or1536 well plate.

In order to achieve a high statistical probability that each reactionarea or well in the microtiter plate contains either a single individualfungal spore or no fungal spore, the resuspended plurality of spores canbe diluted. In one embodiment, the dilution is such that the suspensionof spores is at a concentration whereby the probability that a dispensedor discrete volume of the suspension contains either one or no sporefollows a Poisson Distribution. Further to this embodiment, greater than90% of the wells will contain no spores and thus be empty. Of theremaining wells, greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 99.5% will have a single cell, and less than 2%, 1%, 0.5% or0% will have 2 or more cells. Dispensing of the suspension of spores canbe accomplished using any of the liquid handling devices provided herein(see Table 3) and/or known in the art.

In another embodiment, following resuspension and dilution of theplurality of spores, discrete volumes of the suspension are screened forthe presence or absence of single individual fungal spores in thediscrete volume. Further to this embodiment, if a discrete volume of thesuspension contains only a single individual fungal spore, said discretevolume is distributed, placed or dispensed into a well or reaction area.The screening can be performed using any of the single cell/sporedispensing devices provided herein (see Table 3) and/or known in theart. In one embodiment, the device optically identifies single cells orspores. The device can be a FACS device, a CellenONE device, a CytenaSingle Cell Printer or a Berkeley Lights Beacon device. Prior toresuspension and dilution, the plurality of individual fungal spores canbe picked or isolated using any of the devices provided herein (seeTable 3) and/or known in the art. The resuspension and dilution of thespores can be accomplished using any of the devices provided herein (seeTable 3) and/or known in the art.

Following the screening for the presence or absence of single individualfungal spores in the discrete volume, the methods described hereinfordistributing or dispensing individual spores to the wells or reactionareas of a substrate comprising wells or reaction areas can result in atleast 70%, 75%, 80%, 85%, 90%, 95%, 99 or 99.5% of the wells or reactionareas in the substrate containing a single individual viable spore froma plurality of spores. Using the methods described herein fordistributing individual spores to the wells or reaction areas of asubstrate comprising wells or reaction areas can result in greater than70%, 75%, 80%, 85%, 90%, 95%, 99 or 99.5% of the wells or reaction areasin the substrate containing a single individual viable spore from aplurality of spores. Using the methods described herein for distributingindividual spores to the wells or reaction areas of a substratecomprising wells or reaction areas can result in substantially all ofthe wells or reaction areas in the substrate containing a singleindividual viable spore from a plurality of spores. Using the methodsdescribed herein for distributing individual spores to the wells orreaction areas of a substrate comprising wells or reaction areas canresult in all or 100% of the wells or reaction areas in the substratecontaining a single individual viable spore from a plurality of spores.Using the methods described herein for distributing individual spores tothe wells or reaction areas of a substrate comprising wells or reactionareas can result in a statistical probability that greater than or atleast 70%, 75%, 80%, 85%, 90%, 95%, 99 or 99.5% of the wells in themicrotiter plate contain a single individual viable spore. Using themethods described herein for distributing individual spores to the wellsor reaction areas of a substrate comprising wells or reaction areas canresult in a statistical probability that all or substantially all of thewells in the microtiter plate contain a single individual viable spore.The substrate can be a microtiter plate. The microtiter plate can be a96 well, 384 well or 1536 well plate.

The plurality of individual fungal spores can be derived from afilamentous fungal strain. The filamentous fungal strain can be selectedfrom Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora(e.g., Myceliophthora thermophila), Mucor, Neurospora, Penicillium,Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus,Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus,Thielavia, Tramates, Tolypocladium, Trichoderma, Verticillium,Volvariella species or teleomorphs, or anamorphs, and synonyms ortaxonomic equivalents thereof. In one embodiment, the filamentous fungalstrain is Aspergillus niger. In another embodiment, the filamentousfungal strain possess a non-mycelium forming phenotype. In yet anotherembodiment, the filamentous fungal strain possesses a non-functionalnon-homologous end joining (NHEJ) pathway. The NHEJ pathway can be madenon-functional by exposing the cell to an antibody, a chemicalinhibitor, a protein inhibitor, a physical inhibitor, a peptideinhibitor, or an anti-sense or RNAi molecule directed against acomponent of the NHEJ pathway.

The liquid used for resuspending the plurality of individual spores canbe culture media or a buffer. Further, the wells or reaction areas cancomprise selective media that can serve to select spores containing aspecific genetic perturbation such that culturing the distributedindividual fungal spores in the reaction areas or wells comprising mediaselective for the genetic variation facilitates identification andselection of colonies derived from an individual spore that containedthe desired genetic perturbation.

Aside from or in addition to employing strategies to increase the numberor percentage of mononuclear protoplasts prior to transformation,strategies can be employed to drive protoplasts (and the coloniesderived therefrom following regeneration of said protoplasts) to beinghomokaryotic post-transformation regardless of whether they are mono- ormulti-nucleate. As provided herein, increasing the number or percentageof protoplasts (and the colonies derived therefrom) that arehomokaryotic for a desired or target gene of interest can entailsubjecting the colonies derived from the transformed protoplast orpopulation of transformed protoplasts to selection and/orcounter-selection based on the presence and/or absence of one or moreselectable markers. The one or more selectable markers can be anyselectable marker or combination of selectable markers as providedherein and the selection and/or counter-selection scheme can any suchscheme as provided herein.

Identification of Homokaryotic Transformants

Homokaryotic transformants produced by the methods provided herein canbe identified through the use of phenotypic screening, sequence-basedscreening or a combination thereof. In other words, phenotypicscreening, sequence-based screening or a combination thereof can be usedto detect the presence or absence of a parental genotype in a colonyderived from a protoplast following transformation of said protoplastwith a construct (e.g., insert DNA fragment). Identification ordetection of homokaryotic transformants can occur before and/orfollowing subjecting said transformants to a selection and/orcounter-selection scheme as provided herein in keeping with theintroduction and/or loss of one or more selectable marker genes.Phenotypic screening can be used to identify a transformant with adiscernable phenotype (change in growth and/or colorimetric change),while sequence-based screening can be used to identify transformantswith or without a discernable phenotype following transformation andintegration of a construct or constructs as provided herein.

Sequence-Based Screening

As described herein, sequence-based screening can be used to determinethe presence or absence of a desired or target construct in atransformant. In this manner, sequence-based sequencing can be used toassess whether or not integration of a desired gene or construct hasoccurred in a specific transformant. Sequence-based screening can beused to determine the percentage of nuclei in a multinucleate cell orpopulation of multinucleate cells that contain a desired gene, mutationor construct. Further, sequence-based screening can be used to determinethe percentage of a population of transformants that has experienced adesired target integration. The construct can be any construct or aplurality of constructs as described herein. In some cases, the resultsof sequence-based screening can be used to select purification schemes(e.g., homokaryotic purification) if the percentage or ratio of nucleicomprising a desired gene, mutation or construct vs. nuclei lacking saiddesired gene, mutation or construct is below a certain threshold.

In general, sequence-based screening can entail isolating transformantsthat may contain a desired mutation or construct. Each transformant maycontain one or a plurality of nuclei such that the one or each of theplurality of the nuclei contain fragments of nucleic acid (e.g., one ormore constructs or genes comprising a mutation) introduced duringtransformation. The transformation can be targeted transformations ofprotoplasts with specific fragments of DNA (e.g., one or more constructsor genes comprising a mutation) as provided herein.

In some cases, following isolation, sequence-based screening entailspropagating the transformants that contain a mixture of nuclei with boththe target gene (introduced construct) and the wild-type or parentalgene on media that impacts the purity of the target gene (i.e.,selective media) or may be completely non-selective for any particularphenotype or trait, thereby generating colonies derived from thetransformants. In one embodiment, each isolated transformant or aportion of a colony derived therefrom is transferred to or placed in awell of a microtiter plate such as, for example, an Omnitray (see FIG.30 and seed plate in FIG. 31) comprising agar wherein the transformantor a portion of a colony derived therefrom sporulate. The microtiterplate can be a 96 well, 384 well or 1536 well microtiter plate.

Following isolation alone or in combination with propagation, nucleicacid (e.g., DNA) can be extracted from the transformant or colonies orspores derived therefrom. As shown in FIG. 30, nucleic acid isolationcan be from spores derived from transformants and can be performed in amicrotiter plate format, and can utilize automated liquid handling.Extraction of the nucleic acid can be performed using any known nucleicacid extraction method known in the art and/or commercially availablekit such as for example Prepman™ (ThermoFischer Scientific). In oneembodiment, nucleic acid extracted from spores derived fromtransformants is performed using a boil prep method that allows foramplification of DNA (see FIG. 32). The boil prep method can include theinoculation of spores into a small amount of growth media. In oneembodiment, the spores are separated into 96 wells in a plate suitablefor PCR wherein each well comprises the small amount of growth media.The spores can be allowed to grow for between 10 and 16 hours, which canhelp the spores discard pigments that may inhibit PCR. Additionally, thegrowth can also facilitate several rounds of nuclear division which canserve to increase the genomic DNA content of each well. Subsequently,the overnight “mini cultures” can then be supplemented with a bufferthat assists in cell lysis as well as stabilizes the DNA that will bereleased during lysis. One example of a suitable buffer can be PrepManUltra (Thermo Fisher). Other examples of suitable buffers can includeTris buffered solutions that contain a small amount of ionic detergent.The min-culture-buffer mixtures can then be heated in a thermocycler to99 degrees C. for any of a range of incubation times of between 15minutes and 1 hour.

Following nucleic acid extraction, sequence-based screening can beperformed to assess the percentage or ratio of target or mutant nucleicomprising an introduced target gene or construct to parent nuclei(i.e., non-transformed nuclei). The sequence-based screening can be anymethod known in the art that can be used to determine or detect thesequence of a nucleic acid. The method used to perform sequence-basedscreening can be selected from nucleic acid sequencing methods orhybridization based assays oe methods. The nucleic acid sequencing assayor technique utilized by the methods provided herein can be a nextgeneration sequencing (NGS) system or assay. The hybridization basedassay for detecting a particular nucleic acid sequence can entail theuse of microarrays or the nCounter system (Nanostring). Prior toconducting sequence-based screening, the extracted nucleic acid can beamplified using PCR with primer pair(s) directed to the target gene.

In embodiments utilizing nucleic acid sequencing methologies, the primerpairs utilized in the PCR can comprise adapter sequences that can besubsequently used in a secondary amplification using coded indexingprimers. Amplicons generated by the secondary amplification reaction canthen be sequenced using multiplex sequencing with sequencing primersdirected to the coded indexed primers. The sequencing can be performedusing any type of sequencing known in the art. In one embodiment, thesequencing is next generation sequencing (NGS). The NGS can be any knownNGS method known in the art such as, for example, Illumina NGS. FIG. 30depicts an embodiment of a workflow whereby transformation, sporulation,nucleic acid extraction and NGS based sequence-based screening isperformed in an automated or semi-automated manner. As shown in FIGS.25, 26, 34 and 35, data from the multiplex sequencing reactions can thenbe used to determine the presence or absence of the target nuclei. Insome cases, the data from the multiplex sequencing reactions can also beused to determine the ratio of parental nuclei to mutant nuclei for atransformant within the target well (see FIGS. 34 and 35). Further tothis embodiment, a standard curve can be generated in order to quantifythe percentage or ratio of parent to mutant nuclei. The standard curvecan be generated by amplifying and sequencing nucleic acid isolated fromstrains containing known ratios of a parent to mutant nuclei such asshown in FIG. 33 and subsequently using the ratio of parent to mutantamplicons that appear in the known ratio to determine an approximationof the purity of a test sample. The strains used to generate thestandard curve can be processed (e.g., isolated, propagated andextracted) in the same set of plates as the test sample.

In one embodiment, sequence-based sequencing is used following selectionand/or counter-selection in order to assess or determine thehomokaryotic status of each transformant. Sequence-based sequencing postselection and/or counter-selection can use multiplex sequencing asdescribed herein and can be automated or semi-automated. Sequence-basedsequencing post selection and/or counter-selection can also utilizegeneration of a standard curve as described herein as means ofdetermining the presence and/or amount (e.g., ratio) a transformant isheterokaryotic.

Use of Sequence-Based Screening to Determine Purity of Transformants

As discussed herein, protoplasts generated from coenocytic host cells(e.g., filamentous fungal host cells) in the methods, systems andworkflows provided herein can be multinucleate. Subsequently,protoplasts transformed with one or more constructs such as thoseprovided herein can contain only a portion or percentage of theirmultiple nuclei with a particular construct or constructs integratedinto their genome. Depending on the nature of the transformedconstructs, colonies derived from the transformed protoplast may notproduce a discernable phenotype due to the presence of the mixedpopulation of nuclei present in the colony. Accordingly, the use ofsequence-based screening can be essential for determining the percentageof the nuclei in a mixed population of nuclei that contain a desiredconstruct or constructs vs. those that do not contain a desiredconstruct or constructs. FIGS. 33-35 show the utility of NGS basedscreening for detecting parental vs. mutant nuclei in coloniescontaining cells with a mixed population of nuclei. In one embodiment,NGS based screening is used to identify transformants or strains derivedtherefrom that contain a desired percentage of nuclei with an introducedconstruct or constructs. The desired percentage can be a thresholdpercentage, whereby transformants or strains derived therefrom at orabove said threshold percentage produce a desired product of interest orlevel thereof. The product of interest can be selected from a productlisted in Table 2. The desired percentage can be 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. The percentage can bedetermined by utilizing a standard curve as described herein.

Phenotypic Screening

As described herein, phenotypic screening can be used in combinationwith sequence-based screening or transformants. In some cases, theresults of sequence-based screening can be used to determinepurification schemes in order to ensure the isolation of homokaryotictransformants. Further, sequence-based screening can be utilizedfollowing phenotypic screening/purification in order to assess if theisolates obtained by phenotypic screening/purification are homokaryotic.

Phenotypic screening of transformants generated using the methods,compositions or systems provided herein can employ the use of one ormore selectable markers. A selectable marker can often encode a geneproduct providing a specific type of resistance foreign to thenon-transformed strain. This can be resistance to heavy metals,antibiotics or biocides in general. Prototrophy can also be a usefulselectable marker of the non-antibiotic variety. Auxotrophic markers cangenerate nutritional deficiencies in the host cells, and genescorrecting those deficiencies can be used for selection.

There is a wide range of selection markers in use in the art and any orall of these can be applied to the methods and systems provided herein.The selectable marker genes for use herein can be auxotrophic markers,prototrophic markers, dominant markers, recessive markers, antibioticresistance markers, catabolic markers, enzymatic markers, fluorescentmarkers, luminescent markers or combinations thereof. Examples of theseinclude, but are not limited to: amdS (acetamide/fluoroacetamide), ble(belomycin-phleomycin resistance), hg (hygromycinR), nat (nourseotricinR), pyrG (uracil/5FOA), niaD (nitrate/chlorate), sutB(sulphate/selenate), eGFP (Green Fluorescent Protein) and all thedifferent color variants, aygA (colorimetric marker), met3(methionine/selenate), pyrE (orotate P-ribosyl transferase), trpC(anthranilate synthase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), mutant acetolactate synthase(sulfonylurea resistance), and neomycin phosphotransferase(aminoglycoside resistance).

In one embodiment, a single selection marker is used for examining thephenotypic effects of a specific mutation of a target gene in the genomeof a coenocytic organism. The coenocytic organism can be a filamentousfungi such as A. niger. The target gene can be a gene involved in abiosynthetic pathway such as, for example, a gene involved in citricacid production. An example of this type of embodiment can be seen inFIG. 39. At the top of FIG. 39, a deletion construct comprising a pyrGgene flanked by sequence homologous to a gene involved in citric acidproduction in A. niger is introduced into a host protoplast comprising aversion of the citric acid production gene comprising a mutation (e.g.,SNP) and lacking a native pyrG gene. Homologous recombination of thepyrG construct generates transformants that can be selected for based onthe presence of pyrG gene as described herein. Further, transformantscan have a deletion phenotype that can be used to inform about the rolesaid mutation plays in the pathway. Alternatively, the bottom of FIG.39, depicts an embodiment where a construct comprising a gene involvedin citric acid production with a specific mutation (e.g., SNP) flankedby sequence homologous to the native pyrG locus in the host protoplastis introduced into the host protoplast. Homologous recombination betweenthe construct and the native pyrG locus can generate transformants thatcan be selected for by growing said transformants on media comprisingFOA. The phenotypic effects of the introduced SNP can then be assessedas described herein.

Another embodiment of the present disclosure entails the use of two ormore selection markers active in filamentous fungi. There is a widerange of combinations of selection markers that can be used and all ofthese can be applied in the selection/counterselection scheme providedherein. For example, the selection/counterselection scheme can utilize acombination of auxotrophic markers, prototrophic markers, dominantmarkers, recessive markers, antibiotic resistance markers, catabolicmarkers, enzymatic markers, fluorescent markers, and luminescentmarkers. A first marker can be used to select in the forward mode (i.e.,if active integration has occurred), while additional markers can beused to select in the reverse mode (i.e., if active integration at theright locus has occurred). Selection/counterselection can be carried outby cotransformation such that a selection marker can be on a separatevector or can be in the same nucleic acid fragment or vector as theendogenous or heterologous gene as described herein.

In one embodiment, the homokaryotic protoplast purification scheme ofthe present disclosure entails co-transforming protoplasts generatedform filamentous fungal host cells with a first construct comprisingsequence for an endogenous gene or heterologous gene and a secondconstruct comprising sequence for a first selectable marker gene suchthat the first construct is directed to a first locus of the protoplastgenome that comprises sequence for a target gene to be removed orinactivated, while the second construct is directed to a second locus ofthe protoplast genome that comprises sequence for a second selectablemarker gene. In one embodiment, the first construct comprises sequencefor an endogenous gene or heterologous gene and the target gene to beremoved or inactivated is for a third selectable marker gene. In aseparate embodiment, the first construct comprises a sequence for anendogenous gene and the target gene to be removed or inactivated is thecopy of the endogenous gene present in the genome of the protoplastprior to transformation. As described herein, the endogenous gene orheterologous gene of the first construct can comprise a mutation (e.g.,SNP) and/or a genetic regulatory or control element (e.g., promoterand/or terminator). The first, second and/or third selectable marker canbe any auxotrophic markers, prototrophic markers, dominant markers,recessive markers, antibiotic resistance markers, catabolic markers,enzymatic markers, fluorescent markers, luminescent markers known in theart and/or described herein. To be directed to a specific locus each ofthe constructs is flanked by nucleotides homologous to the desired locusin the protoplast genome as described herein.

An example of the embodiment where the first construct comprisessequence for an endogenous gene or heterologous gene and the target geneto be removed or inactivated is for a third selectable marker gene isshown in FIG. 23A. In this example, the first construct comprisessequence for an endogenous gene involved in citric acid production infilamentous fungus that comprises a SNP that is integrated into thelocus for the colorimetric selectable marker gene aygA, while the secondconstruct comprises sequence for the auxotrophic marker gene pyrG thatis integrated into the locus for the auxotrophic marker gene met3. Inthis example, the filamentous fungal host cell is pyrG negative oruracil auxotrophic. Accordingly, purification of homokaryotic protoplasttransformants entails growing said transformants on minimal medialacking uracil. As shown in FIG. 23A, homokaryotic transformants willnot only be uracil prototrophs, but will also be pure yellow in color,indicting incorporation of the pyrG gene and removal of the aygA gene.Counterselection and removal of any residual heterokaryotic colonies canbe accomplished by subsequently plating the transformants on minimalmedia (with or without uracil) that contains selenate, wherebytransformants with met3+ nucleic will die in the presence of selenate.Another marker that operates similarly to the met3 gene can be, forexample, the niaA gene encoding nitrate reductase, which can be used inthe selection/counterselection scheme described in this embodiment. Forthe niaA gene, the filamentous fungal host cells can be niaA+, wherebycorrect integration of the second construct generates niaA-progeny whichare resistant to chlorate used during counterselection. In oneembodiment, confirmation of correct integration of the first and/orsecond construct into the protoplast genome is confirmed by sequencingthe genome of the protoplast using such as, for example next generationsequencing (NGS).

An example of the embodiment where the first construct comprises asequence for an endogenous gene and the target gene to be removed orinactivated is the copy of the endogenous gene present in the genome ofthe protoplast prior to transformation is shown in FIG. 24. In thisexample, the first construct comprises sequence for an endogenous geneinvolved in citric acid production in filamentous fungus that comprisesa SNP that is integrated into the locus for said endogenous gene lackingsaid SNP, while the second construct comprises sequence for theauxotrophic marker gene pyrG that is integrated into the locus for thecolorimetric marker gene aygA. In this example, the filamentous fungalhost cell is pyrG negative or uracil auxotrophic. Accordingly,purification of homokaryotic protoplast transformants entails growingsaid transformants on minimal media lacking uracil. As shown in FIG. 24,homokaryotic transformants will not only be uracil prototrophs, but willalso be pure yellow in color, indicting incorporation of the pyrG geneand removal of the aygA gene. In one embodiment, confirmation of correctintegration of the first and/or second construct into the protoplastgenome is confirmed by sequencing the genome of the protoplast usingsuch as, for example next generation sequencing (NGS). The NGS system ormethod used can be any NGS system or method known in the art such as forexample Illumina NGS.

In one embodiment, the second construct comprises an expression cassettethat encodes a recyclable or reversible marker. The recyclable orreversible marker can be a disruption neo-pyrG-neo expression cassette.The neo-pyrG-neo construct can be co-transformed with the firstconstruct as described in the above embodiments in a ura-strain offilamentous fungal host cell (e.g., A. niger) and homokaryotictransformants can be selected by plating on uracil deficient medium andselecting pure yellow uracil prototrophs as described above.Subsequently, use of pyrG selection can be regenerated by plating saidhomokaryotic transformants on 5-FOA containing medium and selectingtransformants that grow on said 5-FOA medium, which indicates that saidtransformants have undergone an intrachromosomal recombination betweenthe neo repeats that results in excision of the pyrG gene.

In a further embodiment, instead of using co-transformation as providedherein, the homokaryotic protoplast purification scheme of the presentdisclosure entails transforming protoplasts generated form filamentousfungal host cells with a deletion construct comprising sequence for aspecific gene such that the construct is directed to a desired locus ofthe protoplast genome that comprises sequence for a target gene to beremoved or inactivated. To be directed to a specific locus theconstructs is flanked by nucleotides homologous to the desired locus inthe protoplast genome as described herein. Use of this type ofconstruct/transformation can be used to provide information on the rolea particular gene plays in a particular biochemical pathway. In oneembodiment, confirmation of correct integration of the deletionconstruct into the protoplast genome is confirmed by sequencing thegenome of the protoplast using such as, for example next generationsequencing (NGS). The NGS system or method used can be any NGS system ormethod known in the art such as for example Illumina NGS. Examples ofthis embodiment are shown in FIG. 39. In one case, the filamentousfungal host cell is pyrG negative and the deletion construct comprises aselectable marker gene (e.g., pyrG gene in FIG. 39), while the targetgene is a SNP. Accordingly, purification of homokaryotic protoplasttransformants entails growing said transformants on minimal medialacking uracil. In another case, the filamentous fungal host cell ispyrG positive and the deletion construct comprises a SNP, while thetarget gene is a selectable marker gene (e.g., pyrG gene in FIG. 39).Accordingly, purification of homokaryotic protoplast transformantsentails growing said transformants on minimal media comprising FOA.

In yet another embodiment, a mutated gene (e.g., a SNP) is integratedinto a target locus in the genome of a coenocytic organism (e.g.,filamentous fungi such as A. niger) via transformation and integrationof multiple portions of the mutated gene such that each of the multipleportions of the mutated gene are present on a separate construct. Eachof the multiple constructs can comprise a unique portion of the mutatedgene plus an overlapping portion of the mutated gene that is alsopresent on one of the other multiple constructs in order to facilitaterecombination of the multiple constructs to produce a functional copy ofthe mutated gene in the organism's genome. To facilitate integration ofeach portion of the mutated gene into the desired locus of the organism,each of the multiple constructs can further comprise nucleotideshomologous to the desired locus in the organism's genome that flank theportion of the mutated gene in the construct. In some cases, the mutatedgene is split across two constructs and is introduced into the organismvia bipartite transformation of the two constructs. One example of thisconcept is depicted in FIG. 36. As shown in the left hand column of FIG.36, the pyrG marker gene is split into two constructs such that each ofthe constructs comprises a unique portion of the pyrG and a portion thatoverlaps with the other construct. Further, each construct furthercomprises sequence homologous to the aygA marker gene in the hostorganism genome such that each of the portion of homologous sequence inthe two construct also contains a SNP. Recombination of the twoconstructs following transformation using any of the methods providedherein results in insertion of a the whole pyrG marker gene comprisingthe two SNPs. Transformants containing the wholly integrated pyrG markergene and transformants who have lost the pyrG marker gene via loop-outcan be detected via selection/counterselection as described herein. Inparticular, loop-outs can be selected by growing the transformants onmedia with FOA.

A further example of bipartite transformation is illustrated in FIG. 37.FIG. 37 depicts an example of a combinatorial SNPSWP in fungi (e.g., A.niger) whereby multiple mutations of a target gene (i.e., aygA gene) areintroduced into a protoplast genome by the integration into the parentalaygA gene of two separate constructs each comprising a mutation and aportion of a split marker gene (divergent pyrG genes) in a singletransformation. As shown in FIG. 37, upon successful recombinationbetween the overlapping portions of the respective pyrG gene containingconstructs and between the homologous portions of the aygA gene in theconstructs and host genome, expression of each of the whole pyrG genescan be controlled via catabolite repression by glucose. Accordingly,transformants can be selected by growing the transformants on glucosesuch that the growth of transformants in which the desired recombinationand integration events have occurred will be favored. Further, loop-outscan be facilitated by growing the transformants on media with FOA. Ascan be understood by one skilled in the art, the concepts depicted inFIG. 37 can be used to introduce combinations of mutations (e.g., SNPs)into a target gene and subsequently test the phenotypic effects of saidcombination. The phenotypic effect can be generation of a desiredproperty or activity of an exogenous protein. The property or activityof interest can mean any physical, physicochemical, chemical,biological, or catalytic property, or any improvement, increase, ordecrease in such a property, associated with the exogenous protein. Thephenotypic effect can also be the production or lack of production ofone or more metabolites. The phenotypic effect can also be increased ordecreased quantities of a protein or metabolite. Further, it iscontemplated that further mutations can be introduced using a similartechnique in order to build strains containing specific combinations ofmutations.

The middle and right hand columns of FIG. 36 illustrate additionalapproaches that can be used in the methods and systems provided hereinfor generating transformants with targeted integration of mutations in atarget gene. In one embodiment, co-transformation of a coenocyticorganism (e.g., filamentous fungi) is performed using a first constructcomprising sequence homologous to a desired locus in the host organismgenome, a target gene with a mutation (e.g., SNP) and a portion ofmarker gene (e.g., pyrG) flanked by a terminator repeat (e.g., directrepeat (DR)) and a second construct comprising an overlapping portion ofthe marker gene on the first construct as well as the remainder of themarker gene flanked by a second terminator repeat (DR) and sequencehomologous to the desired locus in the host organism genome.Transformants comprising successful recombination of the constructs andintegration into the desired locus can be isolated using any of theselection/counterselection schemes provided herein (e.g., aygA basedselection and loss of pyrG counterselection in FIG. 36). The right handcolumn of FIG. 36 depicts an example of integration of a mutation (e.g.,SNP) in a target gene (e.g., aygA) using a loop-in single crossoverevent with a construct comprising a copy of the target gene with amutation and one or more selectable markers (e.g., antibiotic resistancegene (amp^(R)) and auxotrophic marker gene (pyrG)).

Library Generation

A further aspect of the disclosure can include the construction andscreening of fungal mutant libraries, and fungal mutant librariesprepared by the methods disclosed herein. As shown in FIG. 5, the fungallibraries can be incorporated into platform for building fungal strains.The libraries may be obtained by transformation of the fungal hostsaccording to this disclosure with any means of integrativetransformation, using methods known to those skilled in the art. Alibrary of fungi based on the preferred host strains generated using themethods and systems provided herein may be handled and screened fordesired properties or activities of exogenous proteins in miniaturizedand/or high-throughput format screening methods. Property or activity ofinterest can mean any physical, physicochemical, chemical, biological,or catalytic property, or any improvement, increase, or decrease in sucha property, associated with an exogenous protein of a library member.The library may also be screened for metabolites, or for a property oractivity associated with a metabolite, produced as a result of thepresence of exogenous and/or endogenous proteins. The library may alsobe screened for fungi producing increased or decreased quantities ofsuch protein or metabolites.

In one embodiment, the methods and systems provided herein generate aplurality of protoplasts such that each protoplast from the plurality ofprotoplasts is transformed with a single first construct from aplurality of first constructs and a single second construct from aplurality of second constructs. Further to this embodiment, a firstpolynucleotide in each first construct from the plurality of firstconstructs comprises a different mutation and/or genetic control orregulatory element while a second polynucleotide in each secondconstruct from the plurality of second constructs is identical. Themethod further comprises transforming and purifying homokaryotictransformants using selection/counterselection as described herein twoor more times in order to generate a library of filamentous fungal cellssuch that each filamentous fungal cell in the library comprises a firstpolynucleotide with a different mutation and/or genetic control orregulatory element. In one embodiment, the first polynucleotidecomprises sequence for a target filamentous fungal gene or aheterologous gene comprising a mutation such that the iterative processgenerates a library of filamentous fungal cells upon regeneration of theprotoplasts such that each member of the library comprises a targetfilamentous fungal gene or a heterologous gene with a distinct mutation.As described herein, the first polynucleotide can be split between morethan one construct such that each construct can comprise an overlappingportion of the first polynucleotide in order to facilitate homologousrecombination between the constructs when introduced into a hostorganism. Further, each construct comprising an overlapping portion ofthe first polynucleotide can further comprise sequence homologous to adesired locus in the host genome in order to facilitate integration ofthe recombined first polynucleotide into the desired locus. In oneembodiment, the mutation is a SNP and the methods thereby produces aSNPSwap library. In one embodiment, the target filamentous fungal geneis a gene involved in citric acid production and the plurality of firstconstructs is the library of SNPs provided in Table 4. In anotherembodiment, the first polynucleotide comprises sequence for a targetfilamentous fungal gene or a heterologous gene operably linked to agenetic control or regulatory element such that the iterative processdescribed herein generates a library of filamentous fungal cells uponregeneration of the protoplasts such that each member of the librarycomprises a target filamentous fungal gene or a heterologous geneoperably linked to a distinct genetic control or regulatory element. Inone embodiment, the genetic control or regulatory element is a promoterand the methods thereby produces a Promoter or PRO library. In oneembodiment, the genetic control or regulatory element is a terminatorand the methods thereby produces a Terminator or STOP library. Thepromoter and/or terminator sequence can be a promoter or terminatorsequence provided herein and/or known in the art for expression in afilamentous fungal host cells used in the methods and systems providedherein. In one embodiment, the promoter is an inducible promoter.

TABLE 4 SNPs potentially involved in citric acid production in A. niger.SNP in Mutation Sequence Coding Morphological name Location changeOrientation Contig Description Domain Phenotype FungiSNP_01 50669- ~>~680224 chr_1_1 680224 FungiSNP_02 1172974 G > A + chr_1_1 Aromatic Xamino acid aminotransferase and related protein FungiSNP_03 367948 C >T + chr_1_2 FungiSNP_04 549014 C > G − chr_1_2 FungiSNP_05 1330718 G >A + chr_1_2 FungiSNP_06 662258 G> + chr_2_1 Taurine X catabolismdioxygenase TauD/TfdA FungiSNP_07 673547 G > A − chr_2_1 alpha/beta Xhydrolase FungiSNP_08 946654 T> + chr_2_1 FungiSNP_09 641661 T > A −chr_2_2 pseudouridylate X X synthase activity FungiSNP_10 2316591 G >A + chr_2_2 FungiSNP_11 935908 A > G − chr_3_1 Serine/threonine Xprotein kinase FungiSNP_12 205638 T > A + chr_3_2 Transcription X Xfactor FungiSNP_13 268107 T > C + chr_3_3 FungiSNP_14 186943 A > T +chr_3_4 FungiSNP_15 276232 C > T + chr_3_4 FungiSNP_16 1287891 T > C −chr_4_1 Serine/threonine X protein kinase FungiSNP_17 1639965 A > T +chr_4_1 FungiSNP_18 1826343 G > A − chr_4_1 Sensory X X transductionhistidine kinase FungiSNP_19 1358794 C > A + chr_4_2 FungiSNP_20 1466380CTA> + chr_4_2 mannitol- X 1-phosphate 5-dehydrogenase FungiSNP_211700330 C > A − chr_4_2 Tomosyn and X related SNARE- interacting proteinFungiSNP_22 2873296 A > G + chr_4_2 FungiSNP_23 815022 G > A + chr_5_2unknown X function FungiSNP_24 831672 G > A − chr_5_2 Cytochrome c Xheme-binding site FungiSNP_25 1507652 >A + chr_5_2 FungiSNP_26 442488T > C + chr_6_1 FungiSNP_27 93202- ~>~ + chr_6_2 103239 FungiSNP_28972833 A > T + chr_6_2 FungiSNP_29 972932 A> + chr_6_2 FungiSNP_301183094 G> + chr_6_2 Monooxygenase X involved in coenzyme Q (ubiquinone)biosynthesis FungiSNP_31 1701762 T > G + chr_6_2 FungiSNP_32 236406 G >A − chr_7_1 extracellular X unknown protein FungiSNP_33 2350056 A> +chr_7_1 FungiSNP_34 375013 C > T + chr_8_1 FungiSNP_35 1272037 C > T +chr_8_1 FungiSNP_36 281612 T > C + chr_8_2 unknown X functionFungiSNP_37 565087 A > G + chr_8_2 FungiSNP_38 865958 A> + chr_8_2FungiSNP_39 947633 A> + chr_8_2 FungiSNP_40 2482267 G > A chr_8_2Uncharacterized X X conserved coiled-coil protein FungiSNP_41 2486601G> + chr_8_2 Magnesium- X dependent phosphatase FungiSNP_42 2709491 T >C + chr_8_2 FunuiSNP_43 2708043 >A ~ chr_8_2 GTPase- X activatingprotein

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. Changes therein and other uses which areencompassed within the spirit of the disclosure, as defined by the scopeof the claims, will be recognized by those skilled in the art.

A brief table of contents (i.e., Table 5) is provided below solely forthe purpose of assisting the reader. Nothing in this table of contentsis meant to limit the scope of the examples or disclosure of theapplication.

TABLE 5 Table of Contents For Example Section Example # Title BriefDescription 1 HTP Genomic Engineering of filamentous fungi: Describesmethods for generating Generation & Storage of Filamentous Fungal andstoring protoplasts for use in Protoplasts HTP genomic engineeringmethods 2 HTP Genomic Engineering of filamentous fungi: Describes analternative method Alternative Method for Generating Protoplasts forgenerating protoplasts for use in HTP genomic engineering methods 3 HTPGenomic Engineering of filamentous fungi: Describes proof-of-principlefor HTP Demonstration of Co-transformation of co-transformation offilamentous Filamentous Fungal Protoplasts-Proof of fungi Principle 4HTP Genomic Engineering of filamentous fungi: Describes HTP method forproof-of- Demonstration of Co-transformation of principle for usingselection/counter- Filamentous Fungal Protoplasts- Proof of selection infilamentous fungal Principle using colorimetric protoplastsselection/counterselection 5 HTP Genomic Engineering of filamentousfungi: Describes HTP method for SNP Implementation of an HTP SNP LibraryStrain library strain improvement program in Improvement Program toImprove Citric Acid A. niger production in Eukaryote Aspergillus nigerATCC 11414 6 HTP Genomic Engineering of filamentous fungi: Describesusing NGS to detect SNPs Demonstration of the ability of next-generationin filamentous fungi sequencing (NGS) to detect SNPs in filamentousfungi with different genetic backgrounds 7 HTP Genomic Engineering offilamentous fungi: Describes using NGS to detect Demonstration of theability of next-generation SNPSWPs in filamentous fungi sequencing (NGS)to detect SNPSWP in filamentous fungi 8 HTP Genomic Engineering offilamentous fungi: Describes HTP co-transformation of Demonstration ofCo-transformation of filamentous fungi with marker and FilamentousFungal Protoplasts-Additional desired gene mutation split across Proofof Principle multiple constructs 9 Non-homologous end joining (NHEJ) andHR- Describes utilization of an RNA mediated genomic editing offilamentous fungi guided endonuclease to edit a using Cas9 ribonucleicacid protein (RNP) filamentous fungi, e.g. A. niger transformationsenable SNPs, insertions, and indels without direct selection for thedesired edits 10 HR-mediated genomic editing of filamentous Describesutilization of a nucleic acid fungi using Cas9 ribonucleic acid protein(RNP) guided nuclease to edit a filamentous transformation to introducesingle SNP without fungi, e.g. A. niger scrambling PAM site 11Purification of Transformed Fungal Strains into Describes a HTP methodfor fungal Clonal Populations at Scale purification to a uniformgenotype through automated separation of spores following transformation12 HTP Genomic Engineering of filamentous fungi: Describes SNP swapmethod for identification of genes that affect filamentous generatingfilamentous fungal strains fungal morphology with non-mycelium, pelletphenotype in submerged CAP culture 13 HTP Genomic Engineering offilamentous fungi: Describes confirmation genes that confirmation ofrole the identified genes play in play a role in morphology offilamentous fungal morphology filamentous fungal strains in submergedCAP culture by knocking out putative morphologically related genes 14HTP Genomic Engineering of filamentous fungi: Describes a PROSWP librarybeing Demonstration of PROSWP in filamentous utilized in filamentousfungi to fungi by altering filamentous fungal cell control expression ofa putative morphology by altering gene expression morphologicallyrelated gene

Example 1: HTP Genomic Engineering of Filamentous Fungi: Generation &Storage of Filamentous Fungal Protoplasts

Generation of Protoplasts

As shown in FIG. 20A, 100 milliliters of complete media was inoculatedwith 10⁶ conidia/ml of Aspergillus niger and grown overnight at 150 rpmat 30° ˜C. Following the overnight growth. the mycelia were harvested byfiltering the culture through Miracloth. Subsequently, the mycelia wererinsed thoroughly with sterile water. For the experiments described inthe following examples, two strains of A. niger were used, A. nigerstrain 1015 and A. niger strain 11414. Harvested and washed mycelia werethen subjected to enzymatic digestion by with a VinoTaste Pro (VTP)enzymatic cocktail. Features of each strain are depicted in FIG. 43.

For A. niger strain 1015, enzymatic digestion was performed by firstmaking 50 ml of 60 mg/ml of VTP in protoplasting buffer (1.2M magnesiumsulfate, 50 mM phosphate buffer, pH 5). After dissolving the VTP, thebuffer was placed in clean Oakridge tubes and spun at 15,000×g for 15minutes. The solution was then filter sterilized after centrifugation.Once made, some of the harvested mycelia was added to the VTP solutionand the mycelia was digested at 30° C. at 80 rpm for 2˜4 hours. Atvarious intervals during VTP digestion, small samples were examinedunder 400× magnification for the presence of protoplasts (i.e., largeround cells that are larger than conidia and are sensitive to osmoticlysis). When most or all of the mycelia were digested, the culture wasfiltered through sterile Miracloth such that 25 ml of the flow throughcontaining the protoplasts were separated into 1 of 2 50 ml Falcontubes. To each of the 25 ml samples, 5 ml of 0.4M ST buffer (0.4MSorbitol, 100 mM Tris, pH 8) was gently overlaid. The overlaid sampleswere then spun at 800×g for 15 minutes at 4° C. in order to form avisible layer between the ST and digestion buffers. The protoplasts werethen removed with a pipette and mixed gently with 25 ml of ST solution(1.0 M sorbitol, 50 mM Tris, Ph 8.0) and respun at 800×g for 10 minutes.The protoplasts should pellet at the bottom of the tube. The protoplastswere then resuspended in 25 ml of ST solution and collected bycentrifugation at 800×g for 10 minutes.

For A. niger strain 11414, enzymatic digestion was performed by firstmaking 40 ml of 30 mg/ml of VTP in protoplasting buffer (0.6M ammoniumsulfate, 50 mM Maleic Acid, pH 5.5). All of the harvested mycelia wereadded to the VTP solution and the mycelia were digested at 30° C. at 70rpm for 3-4 hours. At various intervals during VTP digestion, smallsamples were examined under 400× magnification for the presence ofprotoplasts. When most or all of the mycelia were digested, the culturewas filtered through sterile Miracloth. The filtrate was then spun at800×g for 10 min at 4° C. to pellet the cells. 25 ml of ST solution(1.0M sorbitol, 50 mM Tris, pH 8.0) was added and the cells wereresuspended and respun. The cells were then washed in 10 ml of STCbuffer (1.0M sorbitol, 50 mM Tris, pH 8.0, 50 mM CaCl₂) and collected bycentrifugation at 800×g for 10 min. The protoplasts (˜10⁸/ml) werecounted and adjusted to be at 1.2×10⁷/ml.

For protoplasts generated from either A. niger strain (i.e., 1015 or11414), following enzymatic digestion, 20% v/v of a 40% PEG solution(40% PEG-4000 in STC buffer)) was added to the protoplasts and mixedgently followed by adding 7% v/v of dimethyl sulfoxide (DMSO) to make a8% PEG/7% DMSO solution. Following resuspension, the protoplasts weredistributed to 96 well (25-50 microliters) microtiter plates using anautomated liquid handler as depicted in FIG. 20A, followed by storage atat least −80° C. prior to transformation.

Example 2: HTP Genomic Engineering of Filamentous Fungi: AlternativeMethod for Generating Protoplasts

As shown in FIG. 27, 500 milliliters of complete media was inoculatedwith 10⁶ conidia/ml of Aspergillus niger and grown overnight at 150 rpmat 30° C. Following the overnight growth, the mycelia were harvested byfiltering the culture through Miracloth. Subsequently, the mycelia wererinsed thoroughly with sterile water. Harvested and washed mycelia werethen subjected to enzymatic digestion by with a VinoTaste Pro (VTP)enzymatic cocktail.

Enzymatic digestion was performed by first making 50 ml of 60 mg/ml ofVTP in protoplasting buffer (1.2M magnesium sulfate, 50 mM phosphatebuffer, pH 5). After dissolving the VTP, the buffer was placed in cleanOakridge tubes and spun at 15,000×g for 15 minutes. The solution wasthen filter sterilized after centrifugation. Once made, some of theharvested mycelia was added to the VTP solution and the mycelia wasdigested at 30° C. at 80 rpm for 2-4 hours. At various intervals duringVTP digestion, small samples were examined under 400× magnification forthe presence of protoplasts (i.e., large round cells that are largerthan conidia and are sensitive to osmotic lysis). When most or all ofthe mycelia were digested, the culture was filtered through sterileMiracloth and the filtrate was collected in a graduated cylinder. Thefiltered protoplasts were transferred to a graduated cylinder and abuffer of lower osmolite concentration (5 ml of 0.4M ST buffer (0.4MSorbitol, 100 mM Tris, pH 8) was gently overlaid. The overlaid sampleswere then spun at 800×g for 15 minutes at 4° C. and protoplasts werethen removed with a pipette and mixed gently with 25 ml of ST solution(1.0 M sorbitol, 50 mM Tris, Ph 8.0) and respun at 800×g for 10 minutes.The protoplasts should pellet at the bottom of the tube. The protoplastswere then resuspended in 25 ml of ST solution and collected bycentrifugation at 800×g for 10 minutes.

Example 3: HTP Genomic Engineering of Filamentous Fungi: Demonstrationof Co-Transformation of Filamentous Fungal Protoplasts-Proof ofPrinciple

Preparation of Targeting DNA

In an effort to provide proof of concept (POC) for the automatedfilamentous fungal transformation and screening method depicted in FIGS.20A-C, the DNA sequence of the Aspergillus niger argB gene was obtainedand the proper reading frame was determined. A set of SNPs were thendesigned such that integration of any of said SNPs into the argB locusof the A. niger genome would result in null mutation of the argB gene.The designs were generated as in silico constructs that predicted a setof oligomers that were used to build the constructs using Gibsonassembly.

Automated Transformation of Protoplasts

Protoplasts derived from A. niger strain 1015 generated and stored in 96well plates (100 microliters protoplast/well) as described in Example 1were then subjected to traditional PEG Calcium mediated transformationsusing automated liquid handlers to combine the SNP DNA constructs withthe protoplast-PEG/DMSO mixtures in the 96 well plates. Morespecifically, to 100 microliters of protoplasts, 1-10 micrograms of theSNP DNA constructs (in a volume of 10 microliters or less) were addedand the mixture was incubated on ice for 15 minutes. To this mixture, 1ml of 40% PEG was added and incubated for 15 minutes for roomtemperature. Subsequently, 10 ml of minimum medium plus 1M sorbitol wasadded and shaken at 80 rpm for 1 hour at 30° C. Following thisincubation, the protoplasts were spun down at 800×g for 5 minutes andthen resuspended in 12 ml of minimal medium containing 1M sorbitol and0.8% agar. The following day, using an additional automated liquidhandling step, the protoplasts were plated on to selective media (i.e.,minimal media+arginine) and non-selective media (i.e., minimal media).Successful transformation of the protoplasts generated with theautomated transformation protocol would be expected to be auxotrophicfor arginine and thus not grow on minimal media lacking arginine due totargeting of the argB gene by the SNP constructs.

As shown in FIG. 21, about 3% of the transformants displayed integrationof the targeting DNA constructs at the correct (i.e., argB) locus asevidenced by lack of growth in the minimal media lacking arginine.Confirmation of integration of the SNP containing constructs at thecorrect locus will be confirmed via next generation sequencing.

Example 4: HTP Genomic Engineering of Filamentous Fungi: Demonstrationof Co-Transformation of Filamentous Fungal Protoplasts—Proof ofPrinciple Using Colorimetric Selection/Counterselection

This example demonstrates an additional proof of principle for theautomated, HTP co-transformation of filamentous fungal cells and furtherdemonstrates the use of selection/counterselection for the isolation ofdesired transformants.

Aspergillus Niger Protoplast Formation and Transformation

A large volume (500 ml) of protoplasts of a eukaryotic fungal strain ofAspergillus niger, ATCC 1015, was generated using a commerciallyavailable enzyme mixture which contains beta-glucanase activity asdescribed in Example 1. The protoplasts were isolated from the enzymemixture by centrifugation and were ultimately re-suspended in a buffercontaining calcium chloride by the method described in Example 1.

The protoplasts were aliquoted and frozen at negative 80 degrees Celsiusin containers containing a suspension of dimethyl sulfoxide andpolyethylene glycol (PEG) as described in Example 1. In someembodiments, the present disclosure teaches that a stock of 96-wellmicrotiter plates containing 25-50 microliters of protoplasts in eachwell can be prepared and frozen in large batches for large scale genomeediting campaigns using this technique.

Traditional PEG Calcium mediated transformations were carried out byautomated liquid handlers, which combined the DNA with theprotoplast-PEG mixtures in the 96 wells. An additional automated liquidhandling step was used to plate the transformation on to selective mediaafter transformation.

Automated Screening of Transformants

As discussed in more detail below, the A. niger cells used in thisexample lacked a functional pyrG gene (i.e., pyrG−) were transformedwith a functional pyrG gene, which permitted transformed cells to growin the absence of Uracil. As shown in FIG. 23A-B, the pyrG gene of thisexample was further designed to incorporate into the location of A.niger's wild type met3 gene, thus incorporating a disruption to thenaturally occurring met3 gene. Disruption of the met3 gene furtherresults in the transformants being methionine auxotrophs, providing asecondary screening method for identifying transformants.

Transformants grown on the selective media without Uracil were isolatedand placed into individual wells of a second microtiter plate. Thetransformants in the second microtiter plate were allowed to grow andsporulate for 2-3 days, before being resuspended in a liquid consistingof water and a small amount of detergent to generate a spore stocksuitable for storage and downstream automated screening.

A small aliquot of each of the aforementioned spore stocks was then usedto inoculate liquid media in a third 96 well PCR plate. These smallcultures are allowed to grow over night in a stationary incubator sothat the yellow-pigment containing spores germinate and form hyphae thatare more amenable to selection, and downstream steps.

Following the culturing step, the hyphae of the third PCR plate werelysed by adding a commercially available buffer and heating the culturesto 99 degrees Celsius for 20 minutes. The plates were then centrifugedto separate the DNA suspension supernatant from the cell/organellepellets. The DNA extractions were then used for PCR analysis to identifycell lines comprising the desired DNA modifications.

Co-Transformation for Integration of SNPs-Design of SNPs

The DNA sequence of the Aspergillus niger gene aygA was obtained and theproper reading frame was determined. Four distinct types of mutationswere designed, which if integrated would result in a null mutation.

The mutations included a single base pair change that incorporates anin-frame stop codon, a small two base pair deletion, a three-base pairintegration, and a larger 100 base pair deletion all of which ifproperly integrated will eliminate aygA activity. Strains lacking aygAactivity have a yellow spore phenotype. The designs were generated as insilico constructs that predicted a set of oligomers that were used tobuild the constructs using Gibson assembly.

Integration of SNPs by Co-Transformation

Using the transformation approach described above, amplicons containingthe small changes were incorporated into the genome of an Aspergillusniger strain 1015. As previously discussed, this strain of Aspergillusniger comprised a non-functional pyrG gene, and was therefore unable togrow in the absence of exogenous uracil. Cells that had successfullyintegrated the pyrG gene were now capable of growth in the absence ofuracil. Of these pyrG+ transformants, isolates that also integrated thesmall mutations in the aygA gene exhibited the yellow spore phenotype.(see FIGS. 22 and 23A). The presence of the mutation was also detectedthrough sequencing of small amplicons that contain the region targetedfor the SNP exchange (FIG. 23B).

Example 5: HTP Genomic Engineering of Filamentous Fungi: Implementationof an HTP SNP Library Strain Improvement Program to Improve Citric AcidProduction in Eukaryote Aspergillus niger ATCC 11414

Example 3 above described the techniques for automating the geneticengineering techniques of the present disclosure in a high throughputmanner. This example applies the techniques described above to thespecific HTP strain improvement of Aspergillus niger strain ATCC11414.

Aspergillus niger is a species of filamentous fungi used for the largescale production of citric acid through fermentation. Multiple strainsof this species have been isolated and shown to have varying capacityfor production of citric and other organic acids. The HTP strainengineering methods of the present disclosure can be used to combinecausative alleles and eliminate detrimental alleles to improve citricacid production.

Identification of a Genetic Design Library for SNPs from Natural A.niger Strain Variants.

A. niger strain ATCC 1015 was identified as a producer of citric acid inthe early twentieth century. An isolate of this strain named ATCC 11414,was later found to exhibit increased citric acid yield over its parent(see FIG. 43). For example, A. niger strain ATCC 1015 on averageproduces 7 grams of citric acid from 140 grams of glucose in mediacontaining ammonium nitrate, but lacking both iron and manganesecations. Isolate strain ATCC 11414 on the other hand, exhibits a 10-foldyield increase (70 grams of citric acid) under the same conditions.Moreover, strain ATCC 11414 spores germinate and grow better in citricacid production media than do spores of strain 1015.

In order to identify potential genetic sources for these phenotypicdifferences, the genomes of both the ATCC 1015 and ATCC 11414 strainswere sequenced and analyzed. The resulting analysis identified 43 SNPsdistinguishing the 1015 and 11414 strains (i.e., Table 4).

Exchanging Causative Alleles

Protoplasts were prepared from strain ATCC 1015 (“base strain”) fortransformation as described in Example 1. Each of the above-identified43 SNPs were then individually introduced into the base strain via thegene editing techniques of the present disclosure (see FIG. 24). EachSNP was co-transformed with the functional pyrG and aygA gene mutationas described above. Transformants that had successful gene targeting tothe aygA locus produced yellow spores (FIG. 24).

Screening for Successful Integration

Transformants containing putative SNPs were isolated and a spore stockwas propagated as stated above. Amplicons that contain the region of DNAcontaining the putative SNP were analyzed by next generation sequencing.Using this approach it is possible to determine successful integrationevents within each transformant even in the presence of the parentalDNA. This capability is essential to determine targeting in fungi whichcan grow as heterokaryons which contain nuclei with differing genotypein the same cell.

Transformants were further validated for presence of the desired SNPchange. The cotransformants that had the yellow spore phenotype alsocontained proper integration of the citric acid SNP in approximately 30%of the isolates (FIGS. 25 and 26).

Next, the created SNP swap microbial strain library will bephenotypically screened in order to identify SNPs beneficial to theproduction of citric acid. The information will be utilized in thecontext of the HTP methods of genomic engineering described herein, toderive an A. niger strain with increased citric acid production.

Example 6: HTP Genomic Engineering of Filamentous Fungi: Demonstrationof the Ability of Next-Generation Sequencing (NGS) to Detect SNPs inFilamentous Fungi with Different Genetic Backgrounds

This example demonstrates an example of how NGS can be used to detecttarget gene mutations in a specific background of the target geneparental strain.

In order to test the sensitivity of sequence based screening vs.phenotypic screening, a pair of strains that differ by a single SNP in atarget gene (or test domain) were mixed at known ratios and grown in96-well microtiter plates. The strains used were the parental strain(pyrG−, met+, (P)) and the mutant strain (pyrG+, met−, (M)). Theparental strain spores appear black or dark in color when grown onminimal media (MM), while the mutant strain spores appear yellow orlight in color when grown on MM. The ratios of P:M tested were 1:0,10:1, 5:1, 1:1, 1:5, 1:10, and 0:1). As shown in FIG. 33, when plated onMM supplemented with uracil (+UU) and devoid of methionine (−met), onlyparental spores (P) grow, whereas only mutant spores (M) grow on MM withmet (+met) and devoid of uracil (−UU). As seen in FIG. 33 and the plateat the top of FIG. 35, when plated on MM that is +UU and +met, both Pand M spores grow such that even the lowest ratio (1:10) of P/M sporesproduced black colonies that were visually identical to the highestratio (10:1) of P/M spores. In other words, a single base pair mutationin the avgA gene (M spores) results in the presence of yellow spores;however, the presence of just a few nuclei containing the parental avgAgene (P spores) resulted in colonies that contain black spores.Accordingly, it was difficult to score colonies with small amounts ofparental spores on non-selective media as harboring the mutant gene viaphenotypic screening. In other words, the presence of the mutation wasmasked by even a small number of copies of the parental gene thereforehighlighting the limitations of phenotypic screening in filamentousfungal hosts.

In order to address the limitations of phenotypic screening, thepresence of a target gene mutation was assessed using NGS sequencing ineach of the wells in the 96 well plate from FIG. 33. Here, the nucleicacid from the pairs of strains that differed by a single SNP in the testdomain used in FIG. 33 were using the boil preparation method describedherein. DNA extraction was performed in a 96-well microtiter plateformat whereby a replicate plate was created from the plate in FIG. 33such that DNA extraction was performed in each well of the replicateplate and the isolated DNA was subsequently transferred to an additionalmicrotiter plate using automated liquid handling system (e.g., AgilentBravo system) in which Illumina based sequencing (NGS sequencing) wasperformed in each well. As can be seen in FIG. 34, NGS sequencing wasable to detect the presence of parental and mutant DNA as well asmixtures thereof, whereby the NGS data clearly showed mixtures of singlebase pair changes in the same sample. Given that the experimentsperformed in both FIGS. 33 and 34 were performed using growth conditionsthat did not force homokaryon status, the data showed that NGSsequencing can be utilized as a quality control step during a strainbuilding process in order to assess the efficiency or frequency oftransformation/co-transformation for a particular construct(s) in aparticular strain under particular growth conditions. In other words,NGS can be used to assess or determine the purity of a particulartransformant. In some cases, NGS can be used to determine ifselection/counterselection is necessary for a particular transformant.

In order to assess the ability for NGS to detect the presence, absenceor percentage of mutant vs. parental target genes following a selectionscheme, the parental, mutant and mixed mutant/parental spores from theexperiment depicted in FIG. 33 were grown under conditions that forcedthe presence of colonies that were homokaryotic for either the mutant orparental genotype. More specifically, some of the mixed populations weregrown on media that favored the parental genotype or nuclei (i.e.,minimal media supplemented with uracil and devoid of methionine), whilesome of the mixed populations were grown on media that favored themutant genotype or nuclei (i.e., minimal media supplemented with uraciland devoid of methionine). As can be seen in FIG. 35, selective mediaforced mixed populations of nuclei to homokaryon status for eitherparental nucleic when grown on minimal media supplemented with uraciland devoid of methionine or mutant nuclei when grown on minimal mediasupplemented with uracil and devoid of methionine). Further, thisforcing of homokaryon status by selection was easily detected by NGS.NGS readily detected populations that were entirely homokaryotic for aspecific type of nuclei as well as mixtures thereof. Accordingly, NGScan be used during a strain build process as provided herein in order toassess the efficacy of a particular selection/counterselection scheme.This can be particularly useful when the introduction of specificmutations does not generate a discernable phenotype or a phenotype thatcan be masked by even low percentages of nuclei containing the parentgenotype. This example also illustrates the utility of NGS as a methodfor screening transformants either alone or in combination withphenotypic screening in order to isolate transformants homokaryotic foran introduced DNA insert or transformants with a threshold percentage ofnucleic harboring the introduce DNA insert. The threshold percentage canbe a percentage whereby said transformant produces a desired level of aproduct. The product can be any product known in the art. The productcan be selected from a product in Table 2. The threshold percentage canbe 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.

Example 7: HTP Genomic Engineering of Filamentous Fungi: Demonstrationof the Ability of Next-Generation Sequencing (NGS) to Detect SNPSWP inFilamentous Fungi

This example demonstrates an example of how NGS can be used to detecttarget gene mutations in a specific background of the target geneparental strain. A general scheme of the entire process for the SNPswapping and screening methods can be found in FIGS. 20B-C.

As shown in FIGS. 38A-C, in order to test the ability of sequencingbased screening using NGS to detect multiple integration, ectopicintegrations and/or the presence of SNPs and non-SNPs in the samenuclei, three different scenarios where specific constructs will beintroduced into the aygA locus of a host A. niger genome via homologousrecombination will be examined. In the first scenario, a pyrG markergene is split between two constructs such that an overlapping portion ofthe pyrG gene is present on both constructs. The first construct furthercomprises sequence homologous to the aygA gene present in the hostgenome is present 5′ to the pyrG gene, while the second constructfurther comprises 3′ to the portion of the pyrG gene, sequencehomologous to a second portion of the host aygA gene. In bothconstructs, the sequence homologous to the aygA gene further comprises amutation (i.e., SNP). The second scenario utilizes two constructssimilar to those in the first scenario but only the second constructcomprises a mutation (i.e., SNP) in the sequence homologous to the aygAgene. Finally, the third scenario utilizes two constructs similar tothose used in the second scenario but neither the first or secondconstruct comprise a mutation (i.e., SNP) in the sequence homologous tothe aygA gene.

The A. niger host will be cultivated, protoplasts of A. niger will begenerated and each of the constructs will be transformed into theprotoplasts using the methods described in Example 4. Followingtransformation, transformants will be phenotypically screened for thepresence of the intact aygA gene (black spores) or loss of the intactaygA gene (yellow spores). Additionally, each of the transformants willbe replicate plated and the DNA from the replicate plate will beextracted and screened using NGS as described in Example 6. The expectedresults for both the phenotypic and sequence-based screening areoutlined in FIGS. 38A-C.

Example 8: HTP Genomic Engineering of Filamentous Fungi: Demonstrationof SNPSWP in Filamentous Fungi Using Split-Marker Loop-Out

This example demonstrates a proof of principle for a SNP SWP method infilamentous fungal cells using a split-marker loop-out procedure asshown in FIGS. 3-4. A general scheme of the entire process for the SNPswapping and screening methods can be found in FIGS. 20B-C.

In Examples 4 and 5, targeted SNPSWP in filamentous fungi was performedby transformation of the host using two linear fragments of DNA. One ofthe fragments contained a selectable marker that allows for theisolation of cells that have taken up the DNA. The second independentfragment contained the single base pair change that was to be tested forinfluence on the desired phenotype. In this Example, two fragments areused; however, these fragments both contain the SNP within a directrepeat of DNA that flanks the marker gene. Each fragment only containsone half of the selectable marker. If the fragments integrateindependently they will not reconstitute the marker gene and notransformants will arise. When the marker recombines to form afunctional gene, it will do so through non-homologous end joining. Whenthis occurs it will be much more likely that the flanking sequences willalso properly integrate at the targeted locus. Once the marker hasintegrated at the locus the direct repeats will provide an unstableintegration that can result in the loss of the marker sequence. Strainsthat have lost the marker will leave behind the desired SNP in theproper position and context relative to the gene. Ultimately, theapproach described in Examples 4 and 5 can be used when the SNP iswithin an essential gene and disruption of the gene may be lethal. Inthe approach of Examples 4 and 5, the targeting efficiency of thecotransformation can vary between 5-15%. In contrast, the method of thisExample can be desirable because the marker is linked to the SNP andtargeting efficiency is near 100%.

To perform the method of this Example, A. niger host cells from a 1015parental strain and a 11414 parental strains were cultivated, convertedto protoplasts, and transformed as described in Example 4 above andshown in the scheme depicted in FIG. 20B-C. In this Example, as shown inFIG. 46A, the protoplasts from each parental strain were co-transformedwith two constructs (“split-marker constructs”), wherein each of the twoconstructs contained an overlapping portion of a selectable marker(i.e., pyrG in FIGS. 45 and 46A) and were flanked by direct repeatsequence comprising a target SNP. The split-marker constructs weregenerated using fusion PCR as depicted in FIGS. 45 and 46A and werequality controlled (QC'd) using a fragment analyzer as shown in FIG.46B. Moreover, each of these constructs contained sequence flanking thedirect repeat portions of each construct in order to direct integrationinto a target SNP in the host cell genome. Correct integration wasassessed by screening the transformants using sequence-based screeningas described herein.

Genetic Engineering Using a Split-Marker Approach

The initial steps of the split marker mediated SNP exchange were asshown in FIG. 20B as steps 1 and 2. In step 2, the transforming DNAconsists of two separate linear fragments that contain non-complementaryhalves of the marker fused to homologous DNA for targeting the SNP tothe proper locus. The transformations were placed onto selective mediaand allowed to grow. Properly complemented strains that have stableintegration of the DNA formed colonies. These colonies were pickedeither by hand or by an automated platform to individual wells in amicrotiter plate which contains 100-200 microliters of selective agarmedia. The picked transformants were allowed to grow and propagatespores as indicated in step 4. The spores of A. niger are uninucleateand are inherently clonal. The transformed strains were purified tohomokaryon (all nuclei in the cell are of identical genotype) by takingsmall numbers of spores and plating them again onto selective media.This process is represented by arrows in FIG. 20B. Repeated reduction ofthe population to small numbers of clonal spores resulted in ahomokaryon in each well. These purified strains in wells were thenplated to media containing a counterselection agent that was toxic tostrains that contain the selectable marker. Strains that took up themarker that is flanked by the direct repeats containing the SNP lost themarkers at a frequency that directly correlated to the size of thedirect repeats. For example, a 1,000 base pair direct repeat is lessstable than a 100 base pair direct repeat. This loop out phase is step 6in FIG. 60. FIG. 60 contains data from a SNPSWP campaign that wasperformed utilizing spilt marker integration and loop out.

Results

In this example over 1200 individual looped out samples were screenedusing NGS. From this set, 119 successful strains were generated andappeared as dots in the upper left corner of FIG. 60 because 100% of theamplicons from that well contained the SNP from the production strain.The samples designated in the circled area contained both amplicons withthe desired SNP and the native base pair at this locus. These strainsmay be made homokaryotic using the spore propagation and passingrepresented in steps 4 and 5 in FIG. 20B. The 119 samples that havepassed QC can be analyzed for their impact on desired strain improvementtraits. The success rate of SNP introduction across various SNPpositions is shown in FIG. 61.

Example 9: Non-Homologous End Joining (NHEJ) and HR-Mediated GenomicEditing of Filamentous Fungi Using Cas9 Ribonucleic Acid Protein (RNP)Transformations Enable SNPs, Insertions, and Indels without DirectSelection for the Desired Edits

This example demonstrates an additional means of facile genomic editingin an NHEJ proficient background, without direct selection for thedesired edit. Such a method may be useful for high throughput genomicediting by enabling rapid creation of genomic edits without selectingfor the edit or looping out a selectable marker.

Two crRNA sequences were designed targeting the AygA gene. Disruption ofthis gene results in a null mutation, creating strains that generateyellow-pigment containing spores, rather than black wildtype spores.Disruption could be enabled by NHEJ-mediated error-prone repair or byproviding a homologous recombination donor that disrupts the gene'stranslation.

Assembly of Cas9 RNPs (FIG. 47). Chemically modified crRNA (see Table 6)and tracrRNA (obtained from Synthego) were brought to 250 pmol/μL innuclease free TE buffer. 5 μL of crRNA (250 μM) and 10 μL tracrRNA (250μM) were mixed with 10 μL of 5× annealing buffer (Synthego) and broughtto 50 μL of H₂O. Samples were annealed by bringing the mixture to 78° C.for 10 minutes and then 37° C. for 30 minutes. Samples were then allowedto come to room temperature over 15 minutes. Cas9 RNPs were made bymixing 1 μL of 3.22 μg/IL EnGen Cas9 (obtained from New England Biolabs)with 1.5 μL of crRNA/tracrRNA complex (25 μM) in 0.7 uL STC buffer (1.0Msorbitol, 50 mM Tris, pH 8.0, 50 mM CaCl₂). This was a ratio of 1.875mol of RNA complex/1 mol Cas9 and a total concentration of Cas9 of 1μg/μL. Complexes were formed by incubation at room temperature for 10minutes.

Transformations were performed by first making a 2 fold dilution of theRNP complex in STC. Then, 1 μL of this complex (containing 0.5 ug Cas9)was mixed with 500 ng of vector containing the AMA1 origin and pyrG fromA. fumigatus up to a total of 10 μL in STC. This RNP mixture was addedand mixed to 100 μL of thawed protoplasts of 1015 (PyrG−) (i.e. 1015Aspergillus niger strain) prepared and frozen as described as inExample 1. After the incubation on ice, 1 mL of room temperature 40% PEG4000 made in STC was then added to the protoplast RNP mixture, which wasvortexed and incubated at room temperature for 15 minutes. Protoplastswere then mixed with 12 mL melted minimal media+1M sorbitol and 0.8%agar and plated on 10 cm petri dishes. After solidifying,transformations were overlayed with an additional 8 mL of the meltedminimal media+1M sorbitol and 0.8% agar. A 10× dilution of transformantswere also plated as above (FIG. 48).

TABLE 6 crRNA protospacer sequences SEQ crRNA crRNA ID NO: nameprotospacer sequence 6 aygA.1 UCAGUCUAUCCGUUUCACGA 7 aygA.3UUCCCACGAAGCGAUCACGG 8 control AUGUGUCAGAGACAACUCAA

For cotransformations with two crRNA/tracrRNA/Cas9 complexes, crRNA andtracrRNA pairs were annealed separately and complexed to Cas9separately. After complexation, equal amounts of Cas9 complexed tocrRNA/tracrRNA were mixed together before transformation.

For homologous recombination experiments, donor was created byamplifying gBlocks via PCR and purification. Purified product (800 ng)was added to the RNP and plasmid and transformed as above. RNP cleavageupregulated DNA repair mechanisms, stimulating HR with the exogenouslyprovided donor. The homologous recombination donor sequences weredesigned to contain a mutated region flanked by 400-500 bp of homologyto the AygA gene around the ayg.1 crRNA protospacer cut site. Sequencesshow the two homologous donors used in this experiment.DJV_03_pyrG_insertion_in_AygA shows pyrG with promoter and terminator(lowercase) flanked by 5′ and 3′ regions of homology (uppercase) to theAygA gene (FIG. 51, SEQ ID NO:9). Regions of homology flank thepredicted cut site of crRNA protospacer AygA.1. DJV_07_4bp_insertion_in_AygA contains a 4 bp insertion (lowercase) flanked by 5′and 3′ regions of homology (uppercase) to the AygA gene (FIG. 52, SEQ IDNO:10).

Phenotypes were determined by: (1) counting colonies and later scoringthe color of conidiating colonies, (2) Picking colonies prior toconidiation and then scoring individual isolated colonies afterconidiation, or (3) isolating individual spores 5-7 days aftertransformation and allowing these spores to germinate and conidiate.FIG. 49A-F shows the results of colony counting and scoring ofphenotypic color changes in conidia; using method 1, it was estimatedthat 35-40% of transformed colonies produced yellow conidia. Genotypeswere determined by inoculating 20 μL yeast mold media with spores fromone of these three methods in a static incubator at 37° C. overnight ina wet chamber. Cultures were lysed by mixing 50 μL prepman ultra samplepreparation reagent and incubating mixtures at 98° C. for 30 minutesfollowed by centrifugation to pellet lysate.

Regions flanking a cut site or cut sites were amplified and sequenced bySanger chemistry (FIG. 49D and FIG. 49E). The resulting sequencedamplicons were used to confirm the presence of NHEJ-mediated indels inyellow conidia. To test for homologous recombination, PCR primers weredesigned such that the forward primer annealed upstream of the genomicregion that is homologous to the repair fragment, while the reverseprimer annealed within the region homologous to the repair fragment.This PCR scheme ensures that only genomic DNA and not an unintegrateddonor could result in a PCR amplicon at the AygA locus. For homologousrecombination assays, spores, sorted via the CellenONE, were phenotypedand subsequently genotyped; pyrG payloads were inserted at an efficiencyof 26-36%, 4 bp payloads were inserted with an efficiency of 86% (FIG.50A-C).

Example 10: HR-Mediated Genomic Editing of Filamentous Fungi Using Cas9Ribonucleic Acid Protein (RNP) Transformation to Introduce Single SNPwithout Scrambling PAM Site

This example demonstrates an additional means of facile genomic editingin an NHEJ proficient background using Cas9 RNP-complex transformationto introduce a single SNP without scrambling or altering the PAM siteand without direct selection for the desired edit. Such a method may beuseful for high throughput genomic editing by enabling rapid creation ofgenomic edits without selecting for the edit or looping out a selectablemarker.

Transformations were performed by mixing 0.2 μg EnGen Cas9 (complexed toan annealed Ayg.1 cr/tracRNA) to 350 ng of a double stranded DNA donorand 200 ng of vector containing the AMA1 origin and pyrG from A.fumigatus, brought to 4 μL in STC buffer. This was added to 40 μL ofprotoplasts and transformed as in Example 9. Double stranded donor DNAencoded for a single nonsense mutation flanked on the 5′ and 3′ side by50 bp of homology to the AygA gene (see FIG. 56A, SEQ ID NO: 24); thedonor was made by annealing two complementary oligos. Individualcolonies were picked, and spores from colonies were used to grow,isolate and PCR amplify genomic DNA. Of 24 colonies isolated, 2contained mixtures of black and yellow spores, 2 contained only yellowspores and only 20 contained black spores. Eight of twenty-foursequences were successfully PCR amplified and were sequenced. Of these,three of eight sequences examined contained the desired mutation withoutadditional mutations (see example wildtype (SEQ ID NO: 25) and mutant(SEQ ID NO: 26) traces in FIG. 56B). The remainder of the sequences werewildtype AygA genes. As a result, this Example demonstrates that twoannealed oligos can create a SNP without altering the PAM or seed regionof the protospacer site.

Example 11: Purification of Transformed Fungal Strains into ClonalPopulations at Scale

Many filamentous fungi have a stage in their life cycle in whichvegetative growth includes a state in which multiple nuclei are presentin individual cells. This has a consequence on the ability togenetically manipulate these organisms. Any genetic changes in onenucleus must be made clonal by purification away from nuclei that do notcontain the desired mutation. One method for separating these nuclei isto allow the organism to go through a stage of asexual reproduction inwhich the resulting spores contain few nuclei, all of the same genotype.By separating individual spores, the strains that are propagated fromthese spores will contain the desired production traits. This exampledescribes a method of isolating single spores, and therefore, clonalfungal strains, containing targeted mutations with high fidelity andhigh throughput. The method allows for the rapid generation andpurification of improved fungal production strains.

Traditionally, spore purification can be performed by streaking sporesonto individual petri plates containing selective media. In this method,a spore suspension can be made from each transformed colony whichcontains a mixture of nuclei within the same mycelia. The sporesuspension can be gathered using a sterile loop and then spread asquadrants on the agar plate so that the spores become dilute with eachstreak. The resulting plate should contain some number of spores thatare separated from all of the others and can then be isolated as clonalindividuals.

The method set forth herein eliminates the need to use individual petriplates/dishes for each transformation and facilitates the use ofmicrotiter plates for building strains. Petri dishes are large and notcompatible with automation, therefore limiting to the ability to scaleto high throughput. Most importantly, the present method can yieldclonal populations in >97% of wells, whereas traditional methods knownin the art method may never necessarily result in a clonal population,even after successive passaging (repeated application of the selectionprocess, which can be very inefficient). In the approach detailedherein, individual clonal strains are placed into wells of a microtiterplate for further screening that can facilitate simple integration tohigh-throughput automation. The method can also facilitate the isolationof transformants without the need for colony growth on petri dishes suchthat the entirety of the strain build can occur in a microtiter format.See FIGS. 54A and 54B for the work flow comparisons between thetraditional method known in the art and the method described in thisexample.

Aspergillus niger or a related fungal microbe can produce one or moremetabolites, chemicals, or biologics of interest. Transformationprotocols call for the transformation of A. niger spores with donor DNA.The spores are clonally isolated/deposited into a microtiter plate viathe Poisson distribution or optically based upon single cell dispensing.

Upon transformation, Aspergillus niger is plated and grown tosporulation. The resulting spores are suspended in liquid and diluted.The dilution is performed in one of two ways.

In the first type of dilution, the spores are diluted to a concentrationwhere there is a statistical probability according to a Poissondistribution of only one or no spores existing in the volume dispensed.The spores are then dispensed using an ECHO, BIOSPOT, or other liquidhandling device, into microtiter plates where they can germinate.Generally this approach may generate many empty wells for each well thatcontains a single spore, and ideally very few wells that contain twospores.

In the second type of dilution, the spores are diluted to aconcentration where they can be optically distinguished as single cells.The concentration can be different depending on the instrument used fordispensing. After optical verification that a single cell exists in adroplet, that droplet is dispensed into a microtiter plate. If multiplespores, or none, are in the dispensed volume, they can be put into wastecollection or re-aspirated. Compared to the Poisson distributionapproach (first type of dilution above), it can be expected that eachwell of the output data will have a single cell in it, with far fewerempty or double spore wells.

Instruments for the second type of dilution can include (1) theCellenONE instrument, which uses microfluidics combined with optics tovisually verify that only a single spore are dispensed into a microtiterplate, where they can germinate; (2) the Berkeley Lights Beaconinstrument, which operates by flushing cells or spores into amicrochannel, and then uses a laser to push individual cells or sporesinto micro-holding pens where they can grown and replicate; (3) a FACSinstrument, which uses a microfluidic flow channel to move individualcells past a optical sensor that can detect fluorescence, and can sortcells to output wells based on their fluorescence signal. Unfortunately,currently the only FACS machines on the market are limited to eithersorting cells from a single source to multiple destinations, or aredesigned to select from many sources and sort to a single destination.Should a machine be developed that can easily sort cells from manysources to many destinations, it would be appropriate for thehigh-throughput use case described in this example, with the finalrequirement that the cells being sorted need to be fluorescently active(either naturally, or through genetic engineering); (4) a Cytenainstrument, which operates using similar optical and microfluidictechnology as the CellenONE instrument, but it is not compatible withhigh-throughput/plate-based inputs. It uses a disposable cartridge tohold source liquid, which must be manually bolted to the machine, givingit similar throughput limitations as a FACS machine. The CellenONE canprint single spores with high fidelity, see FIG. 55A-55C.

Example 12: HTP Genomic Engineering of Filamentous Fungi: Identificationof Genes that Affect Filamentous Fungal Morphology

This example demonstrates the use of SNP Swap libraries in a SNPSWAPmethod in the filamentous fungi, Aspergillus niger, in order to identifygenes that play a role in controlling fungal cell morphology. Inparticular, this example describes the identification of a group ofgenes that confer a non-mycelium forming, pellet-like morphologicalphenotype in A. niger mutant strains, where the cells maintain atighter, less elongated phenotype with each cell having multiple tipswhen grown in submerged cultures. This type of growth can be favorableto stirred tank fermentation.

Aspergillus niger is a species of filamentous fungi used for the largescale production of citric acid through fermentation. Multiple strainsof this species have been isolated and shown to have varying capacityfor production of citric acid and other organic acids. The A. nigerstrain ATCC 1015 was identified as a producer of citric acid in theearly twentieth century. An isolate of this strain named ATCC 11414, waslater found to exhibit increased citric acid yield over its parent. Forexample, A. niger strain ATCC 1015 on average produces 7 grams of citricacid from 140 grams of glucose in media containing ammonium nitrate, butlacking both iron and manganese cations. Isolate strain ATCC 11414 onthe other hand, exhibits a 10-fold yield increase (70 grams of citricacid) under the same conditions. Moreover, strain ATCC 11414 sporesgerminate and grow better in citric acid production media than do sporesof strain 1015.

In order to identify potential genetic sources for these phenotypicdifferences, the genomes of both the ATCC 1015 and ATCC 11414 strainswere sequenced and analyzed. The resulting analysis identified 43 SNPsdistinguishing the 1015 and 11414 strains (see Table 4). Of these 43SNPs, 18 were found to be in the coding domains of their respectivegenes (see Table 4).

In order to identify genes that play a potential role in controlling themorphology/growth of filamentous fungi under different cultureconditions, the 43 SNPs from Table 4 were used in a SNP swap process asdescribed herein in order to systematically introduce each individualSNP from Table 4 into the base 1015 strain and examine phenotypedifferences from a morphological standpoint between resulting parent andmutant strains. Conversely, the same type of process was performed inthe 11414 production strain, whereby each of the SNPs from Table 4already present in the genome of 11414 was systemically replaced withwild-type versions of each gene and any resulting difference inmorphology between the parent and mutant strains were noted.

Constructs for Transforming Protoplasts

In this Example, each strain (i.e., 1015 and 11414) was co-transformedwith two constructs (“split-marker constructs”), wherein each of the twoconstructs contained an overlapping portion of a selectable marker(i.e., pyrG in FIGS. 45 and 46A) and were flanked by direct repeatsequence as shown in FIGS. 45 and 46A. The split-marker constructs weregenerated using fusion PCR and were quality controlled (QC'd) using afragment analyzer as shown in FIG. 46B. Moreover, each of theseconstructs further comprised sequence flanking the direct repeatportions of each construct in order to direct integration in the hostcell genome at the respective target gene for each SNP from Table 4. Forthe 1015 base strain protoplasts, the direct repeats in the splitconstructs comprised one of the SNPs from Table 4 (see FIG. 3). Incontrast, for the 11414 production strain protoplasts, the directrepeats did not comprise a SNP from Table 4.

The A. niger base strain 1015 and production strain 11414 werecultivated, converted to protoplasts, transformed and screened asdescribed herein. In summary, each of these steps were as follows:

Generation of Protoplasts

500 milliliters of complete media was inoculated with 10⁶ conidia/ml andgrown overnight at 150 rpm at 30° C. for both the A. niger 1015 basestrain and A. niger 11414 production strain. Following the overnightgrowth, the mycelia were harvested by filtering each culture throughMiracloth. Subsequently, the mycelia were rinsed thoroughly with sterilewater. Harvested and washed mycelia from both strains were then eachseparately subjected to enzymatic digestion with a VinoTaste Pro (VTP)enzymatic cocktail.

Enzymatic digestion of the mycelia for both strains was performed byfirst making 50 ml of 60 mg/ml of VTP in protoplasting buffer (1.2Mmagnesium sulfate, 50 mM phosphate buffer, pH 5). After dissolving theVTP, the buffer was placed in clean Oakridge tubes and spun at 15,000×gfor 15 minutes. The solution was then filter sterilized aftercentrifugation. Once made, some of the harvested mycelia was added tothe VTP solution and the mycelia was digested at 30° C. at 80 rpm for˜2-4 hours. At various intervals during VTP digestion, small sampleswere examined under 400× magnification for the presence of protoplasts(i.e., large round cells that are larger than conidia and are sensitiveto osmotic lysis). When most or all of the mycelia for each strain weredigested, the culture from each strain was filtered through sterileMiracloth and the filtrates were collected in a graduated cylinder. Thefiltered protoplasts were transferred to a graduated cylinder and abuffer of lower osmolite concentration (5 ml of 0.4M ST buffer (0.4MSorbitol, 100 mM Tris, pH 8) was gently overlaid. The overlaid sampleswere then spun at 800×g for 15 minutes at 4° C. and protoplasts werethen removed with a pipette and mixed gently with 25 ml of ST solution(1.0 M sorbitol, 50 mM Tris, Ph 8.0) and respun at 800×g for 10 minutes.The protoplasts should pellet at the bottom of the tube. The protoplastsfrom each strain were then each separately resuspended in 25 ml of STsolution and collected by centrifugation at 800×g for 10 minutes.

Transformation of Protoplasts

Following centrifugation, the protoplasts from both strains wereultimately re-suspended in a buffer containing calcium chloride.Subsequently, protoplasts from both strains were subjected totraditional PEG Calcium mediated transformations using automated liquidhandlers, which combined the DNA from the split constructs describedabove with the protoplast-PEG mixtures in the 96 wells.

Screening for Transformants

As described above, the split marker constructs utilized in this Examplecontained direct repeats flanking the pyrG marker gene, which weresubsequently used for looping out the marker gene. As a result, strainscontaining the loop out construct were counter selected for deletion ofthe selection region (e.g., see FIG. 45 and FIG. 4; absence of pyrGgene). Correct integration was further assessed by sequence-basedscreening as described herein. Further, the mutant strains were screenedusing NGS in order to assess the homokaryotic nature of thetransformants as provided herein. Homokaryotic or substantiallyhomokaryotic mutant strains were plated on minimal media with (see FIGS.62 and 63) or without (see FIG. 64) various supplements in order toassess said strains ability to grow under low pH (FIG. 62) or osmoticstress (FIG. 63) or sporulate (FIG. 64). In addition, the mutant strainswere grown as submerged cultures in CAP media in order to assess theirphenotype in submerged production media.

Results

Individual integration of 4 of the SNPs shared between Table 4 into thebase A. niger strain 1015, generated a morphological phenotype. Inparticular, integration of FungiSNP_9 (SEQ ID NO: 11), FungiSNP_12 (SEQID NO: 12), FungiSNP_18 (SEQ ID NO: 13) or FungiSNP_40 (SEQ ID NO: 14)into the 1015 genome generated mutant strains produced a non-mycelium,pellet morphology when grown as a submerged culture in CAP media.

The role of the genes containing the 4 SNPs in affecting fungalmorphology was further demonstrated in the wave down experiments,whereby removal of each of these 4 SNPs rescued the observedmorphological phenotypes. The sequences of the 4 SNPs can be found inthe attached sequence listing, while their putative or known proteinfunction can be found in Table 4.

As shown in FIG. 62, strains that contain the Base SNP18 grow faster onlow pH media. The presence of FungiSNP_18 from the production strain(11414) in the base strain (i.e., Base snp18^(prod) in FIG. 62) reducedradial growth of the resultant colony on pH2 media as compared to thebase (i.e., Base from FIG. 62). In contrast, the presence of thewild-type version of FungiSNP_18 from the base strain in the productionstrain (i.e., Production SNP18Ba in FIG. 62) allowed for radial growthin said strain as compared to the Base and Production strains from FIG.62. Further, it seems that other SNPs present in the production strainalso contribute to lower radial growth (see Production in smaller thansnp18^(prod) in FIG. 62).

As shown in FIG. 63, strains that contain the base SNP18 (i.e.,wild-type version of FungiSNP_18) grow faster on media which provideosmotic stress. The presence of FungiSNP_18 from the production strain(11414) in the base strain (i.e., Base snp18^(prod) in FIG. 63) reducedradial growth of the resultant colony under osmotic stress as comparedto the base (i.e., Base from FIG. 63). In contrast, the presence of thewild-type version of FungiSNP_18 from the base strain in the productionstrain (i.e., Production SNP18^(Base) in FIG. 63) allowed for radialgrowth in said strain as compared to the Base and Production strainsfrom FIG. 63. Further, it seems that other SNPs present in theproduction strain also contribute to lower radial growth (see Productionin smaller than Base snp18^(prod) in FIG. 63).

Interestingly, base strains containing each of FungiSNP_9, FungiSNP_12,or FungiSNP_40 grew normally and sporulated normally when not grown insubmerged cultures (e.g., on plates). Expressing FungiSNP_18 in the basestrain (i.e., 1015) did show an effect on radial growth rate (reduced)and sporulation as shown in FIG. 64.

Example 13: HTP Genomic Engineering of Filamentous Fungi: Confirmationof Role the Identified Genes Play in Filamentous FungalMorphology-Deletion of the Identified Morphological Control Genes

This example demonstrates confirmation of the role of the 4 genesidentified in Example 12 as playing a role in fungal morphology. Inparticular, this example describes knocking out or deleting each of the4 genes using HTP methods as described herein in A. niger strains 1015and 11414.

The A. niger base strain 1015 and production strain 11414 werecultivated, converted to protoplasts, transformed and screened asdescribed in Example 12.

Constructs for Transforming Protoplasts

In this Example, protoplasts from each strain (i.e., 1015 and 11414)were transformed with a series of single constructs whereby eachconstruct in the series contained a selectable marker gene (i.e., pyrG)flanked by sequence complementary to genomic sequence flanking one ofthe 4 genes of interest identified in Example 12 in order to directintegration of the marker gene into the host cell genome. As shown inFIG. 39, integration of the marker gene into the locus of one of the 4genes (one of the 4 wild-type genes in the 1015 strain and one the of 4SNPs in the 11414 strain) essentially served to remove said wildtypegene or SNP containing gene from the locus of the respective strain.

Following growth, the mutant strains were screened using NGS in order toassess the homokaryotic nature of the transformants as provided herein.Homokaryotic or substantially homokaryotic mutant strains were plated onmedia in order to assess said strains ability to sporulate or grown assubmerged cultures in CAP media in order to assess their phenotype insubmerged production media.

Results

Removal of each of the 4 genes from the base 1015 strain as well as the11414 production strain confirmed the results from Example 12 in thateach of said 4 genes clearly play a role in affecting fungal morphology.In particular, as in Example 12, removal of the non-SNP containingversion of the gene containing FungiSNP_18 in the 1015 strain or thegene containing FungiSNP_18 in the 11414 strain, produced the moststriking phenotype whereby under submerged culture conditions, saidstrains had a pellet like morphology. Further, as shown in FIG. 65,deletion of FungiSNP18 and FungiSNP40 genes resulted in a tightmorphology under all conditions. This data may indicate that the SNPsare not loss of function mutations given that the deletion phenotypesare more pronounced (stronger impact on morphology) than the SNPsthemselves. Thus, it seems that altering the expression of these genesmay impact morphology in a manner that is desirable for growth infermenters.

Interestingly, deletion of the non-SNP containing version of the genecontaining FungiSNP_18 in the 1015 strain produced a negativesporulation phenotype in the resultant variant 1015 strain such thatsaid variant 1015 strain lost the ability to sporulate (see FIG. 66).This loss of sporulation was not observed in the 11414 strain in whichthe FungiSNP_18 gene was removed. Given that the genetic backgrounds ofthe 11414 and 1015 strains are identical aside from the SNPs present inTable 4, this suggested that the presence of one, all or somecombination of the SNPs from Table 4 in the 11414 genetic background isenough to rescue the negative sporulation phenotype produced whenFungiSNP_18 is removed. Put another way, there are other mutations(SNPs) that act epistatically to maintain sporulation in the productionstrain in the absence of SNP18 activity.

It should be noted that the loss of sporulation was not observed ineither the variant 11414 or 1015 strains produced by removingFungiSNP_9, FungiSNP_12 or FungiSNP_40 or their non-SNP containingversions, respectively.

It should be further noted that the observed morphological phenotypesunder submerged culture conditions in this Example were more strikingthan in Example 12 for each of the 4 genes, which could be due to theexperimental design whereby successful transformants essentiallydisplayed a deletion phenotype. Moreover, the phenotypes in the 11414strain were also more pronounced which could be due to contributions tothe phenotype by one or more of the other SNPs present in this strainvs. the 1015 base strain

Example 14: HTP Genomic Engineering of Filamentous Fungi: AlteringFilamentous Fungal Cell Morphology by Altering Gene Expression

This example serves as a proof of principle for the automated, HTPPROSWP method in filamentous fungal cells by showing the use of anautomated, HTP PROSWP method in filamentous fungal cells in order totest the effects of modulating the expression of the FungiSNP_9,FungiSNP_12, FungiSNP_18 and FungiSNP_40 genes identified from Examples1 and 2 that are thought to play a role in controlling filamentousfungal morphology.

In this Example, the expression of the FungiSNP_18 gene (i.e., SEQ IDNO: 13) identified in Examples 12 and 13 was modulated in both the A.niger 1015 base strain and the A. niger 11414 production strain byreplacing the annotated native promoter with one of the four promotersfrom Table 1 using the PROSWP method described herein. Morespecifically, for each of the strains (i.e., the 1015 parent strain orthe 11414 parent strain) for each FungiSNP, a set of (4) variant ormutant strains were generated, where a 1^(st) variant strain expresses afirst construct comprising said candidate FungiSNP (FungiSNP_9 (SEQ IDNO: 11); _12 (SEQ ID NO: 12); _18 (SEQ ID NO: 13); 40 (SEQ ID NO: 14))gene under the control of the srp8p promoter described in Table 1, a 2ndvariant strain had said candidate FungiSNP gene under the control of theamy8p promoter described in Table 1, a 3rd variant strain had saidcandidate FungiSNP gene under the control of the man8p promoterdescribed in Table 1 and a 4th variant strain had said candidateFungiSNP gene under the control of the mbfAp promoter described inTable 1. Each of the constructs used to generate the variants furthercomprised sequence flanking the candidate FungiSNP gene and promoterthat served to direct integration of the construct into the locus of therespective candidate FungiSNP. A general description of the bipartiteconstruct design and integration scheme used in this Example is shown inFIG. 67.

Following their generation, each construct for each candidate FungiSNPused to generate the (4) variant strains was individually transformedinto protoplasts generated for both the A. niger 1015 base strain aswell as the A. niger 11414 production strain. The protoplasts for bothstrains were cultivated, converted to protoplasts, transformed andscreened to select for substantially homokaryotic protoplasts usingphenotypic and/or sequence-based screening as described in the Examplesabove. Accordingly, the transformation of each individual construct ledto the generation of the 4 variant or mutant strains for each of theparental strains for each candidate FungiSNP as generally depicted inFIG. 40. The morphological phenotype of each of these strains was thenobserved and compared with the morphological phenotype of a mutantstrain comprising the identified gene under the control of the nativepromoter for said gene. An ideal level of expression was then determinedfor each of the identified genes

Results

Overall, promoter swapping for each morphology control gene target(i.e., FungiSNP_9, _12, _18 and 40) with the different promoters fromTable 1 revealed that controlling expression of these genes impactedmorphology (see FIG. 68). The strain containing SNP18 under the weakmanB promoter had tighter colony morphology than strains containingother promoter combinations. The impact of SNP18 control was morepronounced under osmotic stress than under low pH. Further, the straincontaining SNP40 under the weak manB promoter had a drastic effect oncolony morphology than strains containing other promoter combinationsunder all growth conditions tested.

As shown in FIG. 69, promoter swapping of morphology control gene target12 (FungiSNP_12; SEQ ID NO: 12) with the different promoters from Table1 revealed that lower strength promoters resulted in yellow pigment inhyphae and some altered morphology observed at the edge of colonies. Thepresence of the yellow pigment indicated that the variant or mutantstrains were experiencing metabolic stress.

Moreover, promoter swapping of morphology control gene target 18(FungiSNP_18; SEQ ID NO: 13) with the different promoters from Table 1revealed that controlling expression of this gene with the two weakerpromoters impacted morphology (see FIGS. 57, 58 and 70). For example,the strains containing the manB fusion and the amyB fusion retained amultiple tip phenotype, whereas those with higher expression srpB andmbfA lacked the multiple tip phenotype and instead showed abnormalswelling (see FIG. 57). The images in FIG. 58 are of strains grown incitric acid production media at 30° C. for 24 hours. The images in FIG.57 are of parent 11414 strains as well as 11414 strains expressingvarious non-native promoter-FungiSNP_18 fusions grown in citric acidproduction media at 30° C. for 48 hours. When allowed to incubate for168 hours, the strains with higher expression promoters as well as theparent strain control all contained long filamentous hyphae. The strainswith the lower level of expression from the promoter fusion, amyB andmanB, remained pelleted. It should be noted that, as shown in FIG. 70,when driven by weaker promoters, SNP_18 has more severe morphologicalphenotype in the base strain than in the production strain.

Similar to the results of the deletion experiments from Example 13,reduction of the expression of the FungiSNP_18 gene in the 1015 strainresulted in cells that experienced a loss of sporulation as shown inFIG. 59. This loss of sporulation was not observed in the 11414 mutantstrains. Again, given that the genetic backgrounds of the 11414 and 1015strains are identical aside from the SNPs present in Table 4, thissuggested that the presence of one, all or some combination of the SNPsfrom Table 4 in the 11414 genetic background is enough to rescue thenegative sporulation phenotype produced when expression of theFungiSNP_18 is reduced.

TABLE 7 SEQUENCES OF THE DISCLOSURE WITH SEQ ID NO IDENTIFIERS GENEHOMOLOGUES, ORTHOLOGUES OR PARALOGS NAME SOURCE SEQ ID NO: COMMENTSmanBp A. niger 1 Native promoter of manB gene amyBp A. oryzae 2 Nativepromoter of amyB gene srpBp A. niger 3 Native promoter of srpB genembfAp A. niger 4 Native promoter of mbfA gene pyrG A. niger 5 NativepyrG gene aygA.1 crRNA protospacer Artificial 6 sequence avgA.3 crRNAprotospacer Artificial 7 sequence Control crRNA protospacer Artificial 8sequence DJV_03_pyrG_insertion_in_AygA Artificial 9 pyrG with promoterand terminator (lowercase) flanked by 5′ and 3′ regions of homology(uppercase) to the AygA gene DJV_07_4bp_insertion_in_AygA Artificial 104 bp insertion (lowercase) flanked by 5′ and 3′ regions of homology(uppercase) to the AygA gene FungiSNP_9 A. niger 11 FungiSNP_12 A. niger12 FungiSNP_18 A. niger 13 A. niger orthologue of S. cerevisiae SLN1FungiSNP_40 A. niger 14 Ypd1 orthologue A. niger 15 A. niger orthologueof S. cerevisiae Ypd1 Ssk1 orthologue A. niger 16 A. niger orthologue ofS. cerevisiae Ssk1 Skn7 orthologue #1 A. niger 17 A. niger orthologue ofS. cerevisiae Skn7 Skn7 orthologue #2 A. niger 18 A. niger orthologue ofS. cerevisiae Skn7 Ssk2 orthologue A. niger 19 A. niger orthologue of S.cerevisiae Ssk2 Aspergillus 20 sequenced portion of genome with BamHIsite created by mutating EcoRV from FIG. 42 aygA A. niger 21 sequencedportion of aygA gene from FIG. 49D aygA A. niger 22 sequenced portion ofaygA gene with indel mutation from FIG. 49D aygA A. niger 23 sequencedportion of aygA gene from FIG. 49E aygA A. niger 24 portion of aygA genecontaining a nonsense mutation from FIG. 56A aygA A. niger 25 sequencedportion of aygA gene from 56B aygA A. niger 26 sequenced portion of aygAgene containing a nonsense mutation from 56B argB A. niger 27 argB genecontaining a mutation from FIG. 44 argB A. niger 28 sequenced portion ofargB gene from FIG. 44 ARGB A. niger 29 sequenced portion of ARGBprotein from FIG. 44 argB A. niger 30 sequenced portion of argB genecontaining a mutation from FIG. 44 ARGB A. niger 31 sequenced portion ofARGB protein from FIG. 44 ARGB A. niger 32 sequenced portion of ARGBprotein from FIG. 44 argB A. niger 33 sequenced portion of argB genecontaining a mutation from FIG. 44 argB A. niger 34 sequenced portion ofargB gene containing a mutation from FIG. 44 ARGB A. niger 35 sequencedportion of ARGB protein from FIG. 44 AYGA A. niger 36 sequenced portionof AYGA protein from FIG. 56A

NUMBERED EMBODIMENTS OF THE DISCLOSURE

Other subject matter contemplated by the present disclosure is set outin the following numbered embodiments:

1. A method for producing a filamentous fungal strain, the methodcomprising:

a.) providing a plurality of protoplasts, wherein the protoplasts wereprepared from a culture of filamentous fungal cells;

b.) transforming the plurality of protoplasts with a first construct anda second construct, wherein the first construct comprises a firstpolynucleotide flanked on both sides by nucleotides homologous to afirst locus in the genome of the protoplast and the second constructcomprises a second polynucleotide flanked on both sides by nucleotideshomologous to a second locus in the genome of the protoplast, whereintransformation results in integration of the first construct into thefirst locus and the second construct into the second locus by homologousrecombination, wherein at least the second locus is a first selectablemarker gene in the protoplast genome, and wherein the firstpolynucleotide comprises a mutation and/or a genetic control element;c.) purifying homokaryotic transformants by performing selection andcounter-selection; andd.) growing the purified transformants in media conducive toregeneration of the filamentous fungal cells.2. The method of embodiment 1, wherein the first construct is split intoconstruct A and construct B, wherein construct A comprises a firstportion of the first polynucleotide and nucleotides homologous to thefirst locus 5′ to the first portion of the first polynucleotide, andwherein construct B comprises a second portion of the firstpolynucleotide and nucleotides homologous to the first locus 3′ to thesecond portion of the first polynucleotide, wherein the first portionand the second portion of the first polynucleotide comprises overlappingcomplementary sequence.3. The method of embodiment 1 or 2, wherein the second construct issplit into construct A and construct B, wherein construct A comprises afirst portion of the second polynucleotide and nucleotides homologous tothe first locus 5′ to the first portion of the second polynucleotide,and wherein construct B comprises a second portion of the secondpolynucleotide and nucleotides homologous to the first locus 3′ to thesecond portion of the second polynucleotide, wherein the first portionand the second portion of the second polynucleotide comprisesoverlapping complementary sequence.4. The method of any one of the above embodiments, wherein eachprotoplast from the plurality of protoplasts is transformed with asingle first construct from a plurality of first constructs and a singlesecond construct from a plurality of second constructs, wherein thefirst polynucleotide in each first construct from the plurality of firstconstructs comprises a different mutation and/or genetic controlelement; and wherein the second polynucleotide in each second constructfrom the plurality of second constructs is identical.5. The method of embodiment 4, further comprising repeating steps a-d togenerate a library of filamentous fungal cells, wherein each filamentousfungal cell in the library comprises a first polynucleotide with adifferent mutation and/or genetic control element.6. The method of any one of the above embodiments, wherein the firstpolynucleotide encodes a target filamentous fungal gene or aheterologous gene.7. The method of any one of the above embodiments, wherein the mutationis a single nucleotide polymorphism.8. The method of any one of the above embodiments, wherein the geneticcontrol is a promoter sequence and/or a terminator sequence.9. The method of any one of the above embodiments, wherein the geneticcontrol element is a promoter sequence, wherein the promoter sequence isselected from the promoter sequences listed in Table 1.10. The method of any one of the above embodiments, wherein theplurality of protoplasts are distributed in wells of a microtiter plate.11. The method of any one of the above embodiments, wherein steps a-dare performed in wells of a microtiter plate.12. The method of embodiment 10 or 11, wherein the microtiter plate is a96 well, 384 well or 1536 well microtiter plate.13. The method of any one of the above embodiments, wherein thefilamentous fungal cells are from an Achlya, Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium,Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus,Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella,Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthorathermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia,Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, anamorphs, synonyms or taxonomic equivalents thereof.14. The method of any one of the above embodiments, wherein thefilamentous fungal cells are from Aspergillus niger.15. The method of any one of the above embodiments, wherein thefilamentous fungal cells possess a non-mycelium forming phenotype.16. The method of any one of the above embodiments, wherein the fungalcells possess a non-functional non-homologous end joining (NHEJ)pathway.17. The method of embodiment 16, wherein the non-functional NHEJ pathwayis due to exposure of the cell to an antibody, a chemical inhibitor, aprotein inhibitor, a physical inhibitor, a peptide inhibitor, or ananti-sense or RNAi molecule directed against a component of the NHEJpathway.18. The method of embodiment 17, wherein the chemical inhibitor is W-7.19. The method of any one of embodiments 6-18, wherein the first locusis for the target filamentous fungal gene.20. The method of any one of embodiments 1-18, wherein the first locusis for a second selectable marker gene in the protoplast genome.21. The method of embodiment 20, wherein the second selectable markergene is an auxotrophic marker gene, a colorimetric marker gene, or adirectional marker gene.22. The method of any one of the above embodiments, wherein the firstselectable marker gene is an auxotrophic marker gene, a colorimetricmarker gene, or a directional marker gene.23. The method of any one of the above embodiments, wherein the secondpolynucleotide is an auxotrophic marker gene, a directional marker gene,or an antibiotic resistance gene.24. The method of embodiment 21 or 22, wherein the colorimetric markergene is an aygA gene.25. The method of any one of embodiments 21-23, wherein the auxotrophicmarker gene is an argB gene, a trpC gene, a pyrG gene, or a met3 gene.26. The method of any one of embodiments 21-23, wherein the directionalmarker gene is an acetamidase (amdS) gene, a nitrate reductase gene(niaD), or a sulphate permease (Sut B) gene.27. The method of embodiment 23, wherein the antibiotic resistance geneis a ble gene, wherein the ble gene confers resistance to pheomycin.28. The method of embodiment 19, wherein the first selectable markergene is an aygA gene and the second polynucleotide is a pyrG gene.29. The method of any one of embodiments 20-27, wherein the firstselectable marker gene is a met3 gene, the second selectable marker geneis an aygA gene and the second polynucleotide is a pyrG gene.30. The method of any one of the above embodiments, wherein theplurality of protoplasts are prepared by removing cell walls from thefilamentous fungal cells in the culture of filamentous fungal cells;isolating the plurality of protoplasts; and resuspending the isolatedplurality of protoplasts in a mixture comprising dimethyl sulfoxide(DMSO), wherein the final concentration of DMSO is 7% v/v or less.31. The method of embodiment 30, wherein the mixture is stored at atleast −20° C. or −80° C. prior to performing steps a-d.32. The method of any one of embodiments 30-31, wherein the culture isat least 1 liter in volume.33. The method of any one of embodiments 30-31, wherein the culture isgrown for at least 12 hours prior to preparation of the protoplasts.34. The method of any one of embodiments 30-33, wherein the fungalculture is grown under conditions whereby at least 70% of theprotoplasts are smaller and contain fewer nuclei.35. The method of any one of embodiments 30-34, wherein removing thecell walls is performed by enzymatic digestion.36. The method of embodiment 35, wherein the enzymatic digestion isperformed with a mixture of enzymes comprising a beta-glucanase and apolygalacturonase.37. The method of any one of embodiments 30-36, further comprisingadding 40% v/v polyethylene glycol (PEG) to the mixture comprising DMSOprior to storing the protoplasts.38. The method of embodiment 37, wherein the PEG is added to a finalconcentration of 8% v/v or less.39. The method of any one of the above embodiments, wherein steps a-dare automated.40. A method for preparing filamentous fungal cells for storage, themethod comprising: preparing protoplasts from a fungal culturecomprising filamentous fungal cells, wherein the preparing theprotoplasts comprises removing cell walls from the filamentous fungalcells in the fungal culture;isolating the protoplasts; andresuspending the isolated protoplasts in a mixture comprising dimethylsulfoxide (DMSO) at a final concentration of 7% v/v or less.41. The method of embodiment 40, wherein the mixture is stored at atleast −20° C. or −80° C.42. The method of any one of embodiments 40-41, wherein the fungalculture is at least 1 liter in volume.43. The method of any of embodiments 40-42, wherein the fungal cultureis grown for at least 12 hours prior to preparation of the protoplasts.44. The method of any one of embodiments 40-43, wherein the fungalculture is grown under conditions whereby at least 70% of theprotoplasts are smaller and have fewer nuclei.45. The method of any one of embodiments 40-44, wherein removing thecell walls is performed by enzymatic digestion.46. The method of embodiment 45, wherein the enzymatic digestion isperformed with mixture of enzymes comprising a beta-glucanase and apolygalacturonase.47. The method of any one of embodiments 40-46, further comprisingadding 40% v/v polyethylene glycol (PEG) to the mixture comprising DMSOprior to storing the protoplasts.48. The method of embodiment 47, wherein the PEG is added to a finalconcentration of 8% v/v or less.49. The method of any one of embodiments 40-48, further comprisingdistributing the protoplasts into microtiter plates prior to storing theprotoplasts.50. The method of any one of embodiments 40-49, wherein the filamentousfungal cells in the fungal culture possess a non-mycelium formingphenotype.51. The method of any one of embodiments 40-50, wherein the filamentousfungal cells in the fungal culture are selected from Achlya, Acremonium,Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium,Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus,Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella,Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthorathermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia,Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof.52. The method of embodiment 50, wherein the filamentous fungal cells inthe fungal culture are Aspergillus niger or teleomorphs or anamorphsthereof.53. A system for generating a fungal production strain, the systemcomprising:one or more processors; andone or more memories operatively coupled to at least one of the one ormore processors andhaving instructions stored thereon that, when executed by at least oneof the one or more processors, cause the system to:a.) transform a plurality of protoplasts derived from culture offilamentous fungal cells with a first construct and a second construct,wherein the first construct comprises a first polynucleotide flanked onboth sides by nucleotides homologous to a first locus in the genome ofthe protoplast and the second construct comprises a secondpolynucleotide flanked on both sides by nucleotides homologous to asecond locus in the genome of the protoplast, wherein transformationresults in integration of the first construct into the first locus andthe second construct into the second locus by homologous recombination,wherein at least the second locus is a first selectable marker gene inthe protoplast genome, and wherein the first polynucleotide comprises amutation and/or a genetic control element;b.) purify homokaryotic transformants by performing selection andcounter-selection; andc.) grow the purified transformants in media conducive to regenerationof the filamentous fungal cells.54. The method of embodiment 53, wherein the first construct is splitinto construct A and construct B, wherein construct A comprises a firstportion of the first polynucleotide and nucleotides homologous to thefirst locus 5′ to the first portion of the first polynucleotide, andwherein construct B comprises a second portion of the firstpolynucleotide and nucleotides homologous to the first locus 3′ to thesecond portion of the first polynucleotide, wherein the first portionand the second portion of the first polynucleotide comprises overlappingcomplementary sequence.55. The method of embodiment 53 or 54, wherein the second construct issplit into construct A and construct B, wherein construct A comprises afirst portion of the second polynucleotide and nucleotides homologous tothe first locus 5′ to the first portion of the second polynucleotide,and wherein construct B comprises a second portion of the secondpolynucleotide and nucleotides homologous to the first locus 3′ to thesecond portion of the second polynucleotide, wherein the first portionand the second portion of the second polynucleotide comprisesoverlapping complementary sequence.56. The system of any one of embodiments 53-55, wherein each protoplastfrom the plurality of protoplasts is transformed with a single firstconstruct from a plurality of first constructs and a single secondconstruct from a plurality of second constructs, wherein the firstpolynucleotide in each first construct from the plurality of firstconstructs comprises a different mutation and/or genetic controlelement; and wherein the second polynucleotide in each second constructfrom the plurality of second constructs is identical.57. The system of any one of embodiments 53-56, further comprisinginstructions to repeat steps a-c to generate a library of filamentousfungal cells, wherein each filamentous fungal cell in the librarycomprises a first polynucleotide with a different mutation and/orgenetic control element.58. The system of any one of embodiments 53-57, wherein the mutation isa single nucleotide polymorphism.59. The system of any one of embodiments 53-58, wherein the geneticcontrol element is a promoter sequence and/or a terminator sequence.60. The system of any one of embodiments 53-58, wherein the geneticcontrol element is a promoter sequence, wherein the promoter sequence isselected from the promoter sequences listed in Table 1.61. The system of any one of embodiments 53-60, wherein steps a-c areperformed in wells of a microtiter plate.62. The system of embodiment 61, wherein the microtiter plate is a 96well, 384 well or 1536 well microtiter plate.63. The system of embodiments 53-62, wherein the filamentous fungalcells are selected from Achlya, Acremonium, Aspergillus, Aureobasidium,Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus,Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia,Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea,Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora,Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor,Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces,Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma,Verticillium, Volvariella species or teleomorphs, or anamorphs, andsynonyms or taxonomic equivalents thereof.64. The system of embodiments 53-62, wherein the filamentous fungalcells are Aspergillus niger.65. The system of any one of embodiments 53-64, wherein the filamentousfungal cells possess a non-mycelium forming phenotype.66. The system of any one of embodiments 53-65, wherein the fungal cellpossesses a non-functional non-homologous end joining pathway.67. The system of embodiment 66, wherein the NHEJ pathway is madenon-functional by exposing the cell to an antibody, a chemicalinhibitor, a protein inhibitor, a physical inhibitor, a peptideinhibitor, or an anti-sense or RNAi molecule directed against acomponent of the NHEJ pathway.68. The system of embodiment 67, wherein the chemical inhibitor is W-7.69. The system of any of any one of embodiments 53-68, wherein the firstlocus is for the target filamentous fungal gene.70. The system of embodiments 53-68, wherein the first locus is for asecond selectable marker gene in the protoplast genome.71. The system of embodiment 70, wherein the second selectable markergene is selected from an auxotrophic marker gene, a colorimetric markergene or a directional marker gene.72. The system of any of embodiments 53-71, wherein the first selectablemarker gene is selected from an auxotrophic marker gene, a colorimetricmarker gene or a directional marker gene.73. The system of any one of embodiments 53-72, wherein the secondpolynucleotide is selected from an auxotrophic marker gene, adirectional marker gene or an antibiotic resistance gene.74. The system of embodiment 71 or 72, wherein the colorimetric markergene is an aygA gene.75. The system of any one of embodiments 71-73, wherein the auxotrophicmarker gene is selected from an argB gene, a trpC gene, a pyrG gene, ora met3 gene.76. The system of any one of embodiments 71-73, wherein the directionalmarker gene is selected from an acetamidase (amdS) gene, a nitratereductase gene (niaD), or a sulphate permease (Sut B) gene.77. The system of embodiment 73, wherein the antibiotic resistance geneis a ble gene, wherein the ble gene confers resistance to pheomycin.78. The system of embodiment 69, wherein the first selectable markergene is an aygA gene and the second polynucleotide is a pyrG gene.79. The system of any one of embodiments 53-68, wherein the firstselectable marker gene is a met3 gene, the second selectable marker geneis an aygA gene and the second polynucleotide is a pyrG gene.80. The system of any one of embodiments 53-79, wherein the plurality ofprotoplasts are prepared by removing cell walls from the filamentousfungal cells in the culture of filamentous fungal cells; isolating theplurality of protoplasts; and resuspending the isolated plurality ofprotoplasts in a mixture comprising dimethyl sulfoxide (DMSO) at a finalconcentration of 7% v/v or less.81. The system of embodiment 80, wherein the mixture is stored at atleast −20° C. or −80° C. prior to performing steps a-c.82. The system of any one of embodiments 80-81, wherein the culture isat least 1 liter in volume.83. The system of any one of embodiments 80-82, wherein the culture isgrown for at least 12 hours prior to preparation of the protoplasts.84. The system of any one of embodiments 80-83, wherein the fungalculture is grown under conditions whereby at least 70% of theprotoplasts are smaller and have fewer nuclei.85. The system of any one of embodiments 80-83, wherein removing thecell walls is performed by enzymatic digestion.86. The system of embodiment 85, wherein the enzymatic digestion isperformed with mixture of enzymes comprising a beta-glucanase and apolygalacturonase.87. The system of any one of embodiments 53-86, further comprisingadding 40% v/v polyethylene glycol (PEG) to the mixture comprising DMSOprior to storing the protoplasts.88. The system of embodiment 87, wherein the PEG is added to a finalconcentration of 8% v/v or less.89. A high-throughput (HTP) method of genomic engineering to evolve afilamentous fungus to acquire a desired phenotype, comprising:a. perturbing the genomes of an initial plurality of filamentous fungalmicrobes having the same genomic strain background, to thereby create aninitial HTP genetic design filamentous fungal strain library comprisingindividual filamentous fungal strains with unique genetic variations;b. screening and selecting individual strains of the initial HTP geneticdesign filamentous fungal strain library for the desired phenotype;c. providing a subsequent plurality of filamentous fungal microbes thateach comprise a unique combination of genetic variation, said geneticvariation selected from the genetic variation present in at least twoindividual filamentous fungal strains screened in the preceding step, tothereby create a subsequent HTP genetic design filamentous fungal strainlibrary;d. screening and selecting individual filamentous fungal strains of thesubsequent HTP genetic design filamentous fungal strain library for thedesired phenotype; ande. repeating steps c)-d) one or more times, in a linear or non-linearfashion, until an filamentous fungal microbe has acquired the desiredphenotype, wherein each subsequent iteration creates a new HTP geneticdesign filamentous fungal strain library comprising individualfilamentous fungal strains harboring unique genetic variations that area combination of genetic variation selected from amongst at least twoindividual filamentous fungal strains of a preceding HTP genetic designfilamentous fungal strain library.90. The HTP method of genomic engineering according to embodiment 89,wherein the initial HTP genetic design filamentous fungal strain librarycomprises at least one library selected from the group consisting of: apromoter swap microbial strain library, SNP swap microbial strainlibrary, start/stop codon microbial strain library, optimized sequencemicrobial strain library, a terminator swap microbial strain library,and any combination thereof.91. The HTP method of genomic engineering according to embodiment 89,wherein the initial HTP genetic design filamentous fungal strain librarycomprises a promoter swap microbial strain library.92. The HTP method of genomic engineering according to embodiment 89,wherein the initial HTP genetic design filamentous fungal strain librarycomprises a promoter swap microbial strain library that contains atleast one bicistronic design (BCD) regulatory sequence.93. The HTP method of genomic engineering according to embodiment 89,wherein the initial HTP genetic design filamentous fungal strain librarycomprises a SNP swap microbial strain library.94. The HTP method of genomic engineering according to embodiment 89,wherein the initial HTP genetic design filamentous fungal strain librarycomprises a microbial strain library that comprises:a. at least one polynucleotide encoding for a chimeric biosyntheticenzyme, wherein said chimeric biosynthetic enzyme comprises:i. an enzyme involved in a regulatory pathway in filamentous fungal;ii. translationally fused to a DNA binding domain capable of binding aDNA binding site; andb. at least one DNA scaffold sequence that comprises the DNA bindingsite corresponding to the DNA binding domain of the chimericbiosynthetic enzyme.95. The HTP method of genomic engineering according to embodiment 89,wherein the subsequent HTP genetic design filamentous fungal strainlibrary is a full combinatorial strain library derived from the geneticvariations in the initial HTP genetic design filamentous fungal strainlibrary.96. The HTP method of genomic engineering according to embodiment 89,wherein the subsequent HTP genetic design filamentous fungal strainlibrary is a subset of a full combinatorial strain library derived fromthe genetic variations in the initial HTP genetic design filamentousfungal strain library.97. The HTP method of genomic engineering according to embodiment 89,wherein the subsequent HTP genetic design filamentous fungus strainlibrary is a full combinatorial strain library derived from the geneticvariations in a preceding HTP genetic design filamentous fungal strainlibrary.98. The HTP method of genomic engineering according to embodiment 89,wherein the subsequent HTP genetic design filamentous fungal strainlibrary is a subset of a full combinatorial strain library derived fromthe genetic variations in a preceding HTP genetic design filamentousfungal strain library.99. The HTP method of genomic engineering according to embodiment 89,wherein perturbing the genome comprises utilizing at least one methodselected from the group consisting of: random mutagenesis, targetedsequence insertions, targeted sequence deletions, targeted sequencereplacements, and any combination thereof.100. The HTP method of genomic engineering according to embodiment 89,wherein the initial plurality of filamentous fungal microbes compriseunique genetic variations derived from an industrial productionfilamentous fungal strain.101. The HTP method of genomic engineering according to embodiment 89,wherein the initial plurality of filamentous fungal microbes compriseindustrial production strain microbes denoted S1Gen1 and any number ofsubsequent microbial generations derived therefrom denoted SnGenn.102. The HTP method according to embodiment 89, wherein the filamentousfungus is selected from Achlya, Acremonium, Aspergillus, Aureobasidium,Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus,Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia,Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea,Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora,Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor,Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces,Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma,Verticillium, Volvariella species or teleomorphs, or anamorphs, andsynonyms or taxonomic equivalents thereof.103. The HTP method according to embodiment 89, wherein the filamentousfungus is Aspergillus niger.104. A method for generating a SNP swap filamentous fungal strainlibrary, comprising the steps of:a. providing a reference filamentous fungal strain and a secondfilamentous fungal strain, wherein the second filamentous fungal straincomprises a plurality of identified genetic variations selected fromsingle nucleotide polymorphisms, DNA insertions, and DNA deletions,which are not present in the reference filamentous fungal strain; andb. perturbing the genome of either the reference filamentous fungalstrain, or the second filamentous fungal strain, to thereby create aninitial SNP swap filamentous fungal strain library comprising aplurality of individual filamentous fungal strains with unique geneticvariations found within each strain of said plurality of individualstrains, wherein each of said unique genetic variations corresponds to asingle genetic variation selected from the plurality of identifiedgenetic variations between the reference filamentous fungal strain andthe second filamentous fungal strain.105. The method for generating a SNP swap filamentous fungal strainlibrary according to embodiment 104, wherein the genome of the referencefilamentous fungal strain is perturbed to add one or more of theidentified single nucleotide polymorphisms, DNA insertions, or DNAdeletions, which are found in the second filamentous fungal strain.106. The method for generating a SNP swap filamentous fungal strainlibrary according to embodiment 104, wherein the genome of the secondfilamentous fungal strain is perturbed to remove one or more of theidentified single nucleotide polymorphisms, DNA insertions, or DNAdeletions, which are not found in the reference filamentous fungalstrain.107. The method for generating a SNP swap filamentous fungal strainlibrary according to embodiment 104, wherein the resultant plurality ofindividual filamentous fungal strains with unique genetic variations,together comprise a full combinatorial library of all the identifiedgenetic variations between the reference filamentous fungal strain andthe second filamentous fungal strain.108. The method for generating a SNP swap filamentous fungal strainlibrary according to embodiment 104, wherein the resultant plurality ofindividual filamentous fungal strains with unique genetic variations,together comprise a subset of a full combinatorial library of all theidentified genetic variations between the reference filamentous fungalstrain and the second filamentous fungal strain.109. The method for generating a SNP swap filamentous fungal strainlibrary according to embodiment 104, wherein the filamentous fungus isselected from Achlya, Acremonium, Aspergillus, Aureobasidium,Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus,Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia,Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea,Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora,Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor,Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces,Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma,Verticillium, Volvariella species or teleomorphs, or anamorphs, andsynonyms or taxonomic equivalents thereof.110. The method for generating a SNP swap filamentous fungal strainlibrary according to embodiment 104, wherein the filamentous fungus isAspergillus niger.111. A method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain, comprising thesteps of:a. providing a parental lineage filamentous fungal strain and aproduction filamentous fungal strain derived therefrom, wherein theproduction filamentous fungal strain comprises a plurality of identifiedgenetic variations selected from single nucleotide polymorphisms, DNAinsertions, and DNA deletions, not present in the parental lineagestrain;b. perturbing the genome of either the parental lineage filamentousfungal strain, or the production filamentous fungal strain, to create aninitial library of filamentous fungal strains, wherein each strain inthe initial library comprises a unique genetic variation from theplurality of identified genetic variations between the parental lineagefilamentous fungal strain and the production filamentous fungal strain;c. screening and selecting individual strains of the initial library forphenotypic performance improvements over a reference filamentous fungalstrain, thereby identifying unique genetic variations that conferphenotypic performance improvements;d. providing a subsequent plurality of filamentous fungal microbes thateach comprise a combination of unique genetic variations from thegenetic variations present in at least two individual filamentous fungalstrains screened in the preceding step, to thereby create a subsequentlibrary of filamentous fungal strains;e. screening and selecting individual strains of the subsequent libraryfor phenotypic performance improvements over the reference filamentousfungal strain, thereby identifying unique combinations of geneticvariation that confer additional phenotypic performance improvements;andf. repeating steps d)-e) one or more times, in a linear or non-linearfashion, until an filamentous fungal strain exhibits a desired level ofimproved phenotypic performance compared to the phenotypic performanceof the production filamentous fungal strain, wherein each subsequentiteration creates a new library of microbial strains, where each strainin the new library comprises genetic variations that are a combinationof genetic variations selected from amongst at least two individualfilamentous fungal strains of a preceding library.112. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the initial library of filamentous fungalstrains is a full combinatorial library comprising all of the identifiedgenetic variations between the parental lineage filamentous fungalstrain and the production filamentous fungal strain.113. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the initial library of filamentous fungalstrains is a subset of a full combinatorial library comprising a subsetof the identified genetic variations between the parental lineagefilamentous fungal strain and the production filamentous fungal strain.114. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the subsequent library of filamentous fungalstrains is a full combinatorial library of the initial library.115. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the subsequent library of filamentous fungalstrains is a subset of a full combinatorial library of the initiallibrary.116. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the subsequent library of filamentous fungalstrains is a full combinatorial library of a preceding library.117. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the subsequent library of filamentous fungalstrains is a subset of a full combinatorial library of a precedinglibrary.118. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the genome of the parental lineage filamentousfungal strain is perturbed to add one or more of the identified singlenucleotide polymorphisms, DNA insertions, or DNA deletions, which arefound in the production filamentous fungal strain.119. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the genome of the production filamentous fungalstrain is perturbed to remove one or more of the identified singlenucleotide polymorphisms, DNA insertions, or DNA deletions, which arenot found in the parental lineage filamentous fungal strain.120. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein perturbing the genome comprises utilizing atleast one method selected from the group consisting of: randommutagenesis, targeted sequence insertions, targeted sequence deletions,targeted sequence replacements, and combinations thereof.121. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein steps d)-e) are repeated until the phenotypicperformance of an filamentous fungal strain of a subsequent libraryexhibits at least a 10% increase in a measured phenotypic variablecompared to the phenotypic performance of the production filamentousfungal strain.122. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein steps d)-e) are repeated until the phenotypicperformance of an filamentous fungal strain of a subsequent libraryexhibits at least a one-fold increase in a measured phenotypic variablecompared to the phenotypic performance of the production filamentousfungal strain.123. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the improved phenotypic performance of step f)is selected from the group consisting of: volumetric productivity of aproduct of interest, specific productivity of a product of interest,yield of a product of interest, titer of a product of interest, andcombinations thereof.124. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the improved phenotypic performance of step f)is: increased or more efficient production of a product of interest,said product of interest selected from the group consisting of: a smallmolecule, enzyme, peptide, amino acid, organic acid, synthetic compound,fuel, alcohol, primary extracellular metabolite, secondary extracellularmetabolite, intracellular component molecule, and combinations thereof.125. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the identified genetic variations furthercomprise artificial promoter swap genetic variations from a promoterswap library.126. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, further comprising:engineering the genome of at least one microbial strain of either:the initial library of filamentous fungal strains, ora subsequent library of filamentous fungal strains,to comprise one or more promoters from a promoter ladder operably linkedto an endogenous ilamentous fungal target gene.127. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the filamentous fungus is selected from Achlya,Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria,Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium,Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g.,Myceliophthora thermophila), Mucor, Neurospora, Penicillium, Podospora,Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof.128. The method for rehabilitating and improving the phenotypicperformance of a production filamentous fungal strain according toembodiment 111, wherein the filamentous fungus is Aspergillus niger.129. A method for generating a promoter swap filamentous fungal strainlibrary, comprising the steps of:a. providing a plurality of target genes endogenous to a basefilamentous fungal strain, and a promoter ladder, wherein said promoterladder comprises a plurality of promoters exhibiting differentexpression profiles in the base filamentous fungal strain; andb. engineering the genome of the base filamentous fungal strain, tothereby create an initial promoter swap filamentous fungal strainlibrary comprising a plurality of individual filamentous fungal strainswith unique genetic variations found within each strain of saidplurality of individual filamentous fungal strains, wherein each of saidunique genetic variations comprises one or more of the promoters fromthe promoter ladder operably linked to one of the target genesendogenous to the base filamentous fungal strain.130. The method according to embodiment 129, wherein the filamentousfungal strain is selected from Achlya, Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium,Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus,Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella,Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthorathermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia,Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof.131. The method according to embodiment 129, wherein the filamentousfungal strain is an Aspergillus niger strain.132. A promoter swap method for improving the phenotypic performance ofa production filamentous fungal strain, comprising the steps of:a. providing a plurality of target genes endogenous to a basefilamentous fungal strain, and a promoter ladder, wherein said promoterladder comprises a plurality of promoters exhibiting differentexpression profiles in the base filamentous fungal strain;b. engineering the genome of the base filamentous fungal strain, tothereby create an initial promoter swap filamentous fungal strainlibrary comprising a plurality of individual filamentous fungal strainswith unique genetic variations found within each strain of saidplurality of individual filamentous fungal strains, wherein each of saidunique genetic variations comprises one or more of the promoters fromthe promoter ladder operably linked to one of the target genesendogenous to the base filamentous fungal strain;c. screening and selecting individual filamentous fungal strains of theinitial promoter swap filamentous fungal strain library for phenotypicperformance improvements over a reference filamentous fungal strain,thereby identifying unique genetic variations that confer phenotypicperformance improvements;d. providing a subsequent plurality of filamentous fungal microbes thateach comprise a combination of unique genetic variations from thegenetic variations present in at least two individual filamentous fungalstrains screened in the preceding step, to thereby create a subsequentpromoter swap filamentous fungal strain library;e. screening and selecting individual filamentous fungal strains of thesubsequent promoter swap filamentous fungal strain library forphenotypic performance improvements over the reference filamentousfungal strain, thereby identifying unique combinations of geneticvariation that confer additional phenotypic performance improvements;andf. repeating steps d)-e) one or more times, in a linear or non-linearfashion, until an filamentous fungal strain exhibits a desired level ofimproved phenotypic performance compared to the phenotypic performanceof the production filamentous fungal strain, wherein each subsequentiteration creates a new promoter swap filamentous fungal strain libraryof microbial strains, where each strain in the new library comprisesgenetic variations that are a combination of genetic variations selectedfrom amongst at least two individual filamentous fungal strains of apreceding library.133. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein the subsequent promoter swap filamentous fungal strain libraryis a full combinatorial library of the initial promoter swap filamentousfungal strain library.134. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein the subsequent promoter swap filamentous fungal strain libraryis a subset of a full combinatorial library of the initial promoter swapfilamentous fungal strain library.135. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein the subsequent promoter swap filamentous fungal strain libraryis a full combinatorial library of a preceding promoter swap filamentousfungal strain library.136. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein the subsequent promoter swap filamentous fungal strain libraryis a subset of a full combinatorial library of a preceding promoter swapfilamentous fungal strain library.137. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein steps d)-e) are repeated until the phenotypic performance of anfilamentous fungal strain of a subsequent promoter swap filamentousfungal strain library exhibits at least a 10% increase in a measuredphenotypic variable compared to the phenotypic performance of theproduction filamentous fungal strain.138. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein steps d)-e) are repeated until the phenotypic performance of anfilamentous fungal strain of a subsequent promoter swap filamentousfungal strain library exhibits at least a one-fold increase in ameasured phenotypic variable compared to the phenotypic performance ofthe production filamentous fungal strain.139. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein the improved phenotypic performance of step f) is selected fromthe group consisting of: volumetric productivity of a product ofinterest, specific productivity of a product of interest, yield of aproduct of interest, titer of a product of interest, and combinationsthereof.140. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein the improved phenotypic performance of step f) is: increased ormore efficient production of a product of interest, said product ofinterest selected from the group consisting of: a small molecule,enzyme, peptide, amino acid, organic acid, synthetic compound, fuel,alcohol, primary extracellular metabolite, secondary extracellularmetabolite, intracellular component molecule, and combinations thereof.141. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein the filamentous fungal strain is selected from Achlya,Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria,Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium,Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g.,Myceliophthora thermophila), Mucor, Neurospora, Penicillium, Podospora,Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof.142. The promoter swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 132,wherein the filamentous fungal strain is an Aspergillus niger strain.143. A method for generating a terminator swap filamentous fungal strainlibrary, comprising the steps of:a. providing a plurality of target genes endogenous to a basefilamentous fungal strain, and a terminator ladder, wherein saidterminator ladder comprises a plurality of terminators exhibitingdifferent expression profiles in the base filamentous fungal strain; andb. engineering the genome of the base filamentous fungal strain, tothereby create an initial terminator swap filamentous fungal strainlibrary comprising a plurality of individual filamentous fungal strainswith unique genetic variations found within each strain of saidplurality of individual filamentous fungal strains, wherein each of saidunique genetic variations comprises one or more of the terminators fromthe terminator ladder operably linked to one of the target genesendogenous to the base filamentous fungal strain.144. The method according to embodiment 143, wherein the filamentousfungal strain is selected from Achlya, Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium,Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus,Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella,Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthorathermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia,Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof.145. The method according to embodiment 143, wherein the filamentousfungal strain is an Aspergillus niger strain.146. A terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain, comprising the steps of:a. providing a plurality of target genes endogenous to a basefilamentous fungal strain, and a terminator ladder, wherein saidterminator ladder comprises a plurality of terminators exhibitingdifferent expression profiles in the base filamentous fungal strain;b. engineering the genome of the base filamentous fungal strain, tothereby create an initial terminator swap filamentous fungal strainlibrary comprising a plurality of individual filamentous fungal strainswith unique genetic variations found within each strain of saidplurality of individual filamentous fungal strains, wherein each of saidunique genetic variations comprises one or more of the terminators fromthe terminator ladder operably linked to one of the target genesendogenous to the base filamentous fungal strain;c. screening and selecting individual filamentous fungal strains of theinitial terminator swap filamentous fungal strain library for phenotypicperformance improvements over a reference filamentous fungal strain,thereby identifying unique genetic variations that confer phenotypicperformance improvements;d. providing a subsequent plurality of filamentous fungal microbes thateach comprise a combination of unique genetic variations from thegenetic variations present in at least two individual filamentous fungalstrains screened in the preceding step, to thereby create a subsequentterminator swap filamentous fungal strain library;e. screening and selecting individual filamentous fungal strains of thesubsequent terminator swap filamentous fungal strain library forphenotypic performance improvements over the reference filamentousfungal strain, thereby identifying unique combinations of geneticvariation that confer additional phenotypic performance improvements;andf. repeating steps d)-e) one or more times, in a linear or non-linearfashion, until an filamentous fungal strain exhibits a desired level ofimproved phenotypic performance compared to the phenotypic performanceof the production filamentous fungal strain, wherein each subsequentiteration creates a new terminator swap filamentous fungal strainlibrary of microbial strains, where each strain in the new librarycomprises genetic variations that are a combination of geneticvariations selected from amongst at least two individual filamentousfungal strains of a preceding library.147. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein the subsequent terminator swap filamentous fungal strain libraryis a full combinatorial library of the initial terminator swapfilamentous fungal strain library.148. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein the subsequent terminator swap filamentous fungal strain libraryis a subset of a full combinatorial library of the initial terminatorswap filamentous fungal strain library.149. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein the subsequent terminator swap filamentous fungal strain libraryis a full combinatorial library of a preceding terminator swapfilamentous fungal strain library.150. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein the subsequent terminator swap filamentous fungal strain libraryis a subset of a full combinatorial library of a preceding terminatorswap filamentous fungal strain library.151. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein steps d)-e) are repeated until the phenotypic performance of anfilamentous fungal strain of a subsequent terminator swap filamentousfungal strain library exhibits at least a 10% increase in a measuredphenotypic variable compared to the phenotypic performance of theproduction filamentous fungal strain.152. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein steps d)-e) are repeated until the phenotypic performance of anfilamentous fungal strain of a subsequent terminator swap filamentousfungal strain library exhibits at least a one-fold increase in ameasured phenotypic variable compared to the phenotypic performance ofthe production filamentous fungal strain.153. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein the improved phenotypic performance of step f) is selected fromthe group consisting of: volumetric productivity of a product ofinterest, specific productivity of a product of interest, yield of aproduct of interest, titer of a product of interest, and combinationsthereof.154. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein the improved phenotypic performance of step f) is: increased ormore efficient production of a product of interest, said product ofinterest selected from the group consisting of: a small molecule,enzyme, peptide, amino acid, organic acid, synthetic compound, fuel,alcohol, primary extracellular metabolite, secondary extracellularmetabolite, intracellular component molecule, and combinations thereof.155. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein the filamentous fungal strain is selected from Achlya,Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria,Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium,Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g.,Myceliophthora thermophila), Mucor, Neurospora, Penicillium, Podospora,Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof.156. The terminator swap method for improving the phenotypic performanceof a production filamentous fungal strain according to embodiment 146,wherein the filamentous fungal strain is an Aspergillus niger strain.157. A filamentous fungal host cell comprising a promoter operablylinked to an endogenous gene of the host cell, wherein the promoter isheterologous to the endogenous gene, wherein the promoter has a sequenceselected from the group consisting of SEQ ID Nos. 1-4.158. The filamentous fungal host cell of embodiment 157, whereinfilamentous fungal host cell has a desired level of improved phenotypicperformance compared to the phenotypic performance of a referencefilamentous fungal strain without the promoter operably linked to theendogenous gene.159. The filamentous fungal host cell according to embodiment 157,wherein the filamentous fungal host cell is selected from Achlya,Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria,Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium,Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g.,Myceliophthora thermophila), Mucor, Neurospora, Penicillium, Podospora,Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof.160. The filamentous fungal host cell according to embodiment 157,wherein the filamentous fungal host cell is Aspergillus niger.161. A filamentous fungal strain library, wherein each filamentousfungal strain in the library comprises a promoter operably linked to anendogenous gene of the host cell, wherein the promoter is heterologousto the endogenous gene, wherein the promoter has a sequence selectedfrom the group consisting of SEQ ID Nos. 1-4.162. The filamentous fungal strain library according to embodiment 161,wherein the filamentous fungal strain is selected from Achlya,Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria,Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium,Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g.,Myceliophthora thermophila), Mucor, Neurospora, Penicillium, Podospora,Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof.163. The filamentous fungal strain library according to embodiment 161,wherein the filamentous fungal strain is Aspergillus niger.164. A method for isolating clonal populations derived from singlefungal spores, the method comprising:(a) providing a plurality of fungal spores in a liquid suspension,wherein the plurality of fungal spores were derived from a fungalstrain:(b) dispensing a discrete volume of the liquid suspension to anindividual reaction area in a substrate comprising a plurality ofreaction areas, wherein each reaction area in the plurality of reactionareas comprises growth media, wherein the dispensing results in aprobability that at least 75% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores;(c) culturing the dispensed single viable fungal spores in the reactionareas comprising growth media; and(d) selecting clonal populations growing in the reaction areas, therebyisolating clonal populations derived from single fungal spores.165. The method of embodiment 164, further comprising screening thediscrete volumes for the presence or absence of a single fungal spore inthe discrete volumes, wherein only the discrete volumes containing asingle fungal spore are selected for step (b).166. The method of embodiment 165, wherein the dispensing results in aprobability that at least 80% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores.167. The method of embodiment 165, wherein the dispensing results in aprobability that at least 90% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores.168. The method of embodiment 165, wherein the dispensing results in aprobability that at least 95% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores.169. The method of embodiment 165, wherein the dispensing results in aprobability that at least 99% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores.170. The method of embodiment 165, wherein the dispensing results in aprobability that substantially all of the individual reaction areascontain no more than a single viable fungal spore from the plurality offungal spores.171. The method of any one of embodiments 165-170, wherein the screeningthe discrete volumes entails optically distinguishing the presence orabsence of a single fungal spore in the discrete volumes.172. The method of embodiment 171, wherein the screening is performedusing a microfluidic device capable of optically distinguishing thepresence or absence of a single fungal spore in the discrete volumes.173. A method for isolating clonal populations derived from singlefungal spores, the method comprising:(a) providing a plurality of fungal spores in a liquid suspension,wherein the plurality of fungal spores were derived from a fungalstrain;(b) diluting the liquid suspension, wherein the dilution is a limitingdilution;(c) dispensing a discrete volume of the dilution to an individualreaction area in a substrate comprising a plurality of reaction areas,wherein each reaction area in the plurality of reaction areas comprisesgrowth media, wherein the limiting dilution results in a probabilitythat the discrete volume of the dilution dispensed to each reaction areacontains either one or no viable spore follows a Poisson Distribution,whereby greater than 90% of the reaction areas in the plurality ofreaction areas contain no viable spores and greater than 90% of reactionareas that contain one or more viable spores contain only a singleviable spore;(d) culturing the dispensed single viable fungal spores in the reactionareas comprising growth media; and(e) selecting clonal populations growing in the reaction areas, therebyisolating clonal populations derived from single fungal spores.174. The method of any of embodiments 164-173, wherein the reactionareas are present in a microtiter plate.175. The method of embodiment 174, wherein the microtiter plate contains96 wells, 384 wells or 1536 wells.176. The method of any of embodiments 164-175, wherein the fungal strainis a filamentous fungal strain.177. The method of embodiment 176, wherein the filamentous fungal strainis selected from Achlya, Acremonium, Aspergillus, Aureobasidium,Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus,Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia,Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea,Myceliophthora (e.g., Myceliophthora thermophila), Mucor, Neurospora,Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor,Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces,Thermoascus, Thielavia, Tramates, Tolypocladium, Trichoderma,Verticillium, Volvariella species or teleomorphs, or anamorphs, andsynonyms or taxonomic equivalents thereof.178. The method of embodiment 177, wherein the filamentous fungal strainis Aspergillus niger or teleomorphs or anamorphs thereof.179. The method of embodiment 178, wherein the filamentous fungal strainpossess a non-mycelium, pellet morphology.180. The method of embodiment 179, wherein the filamentous fungal strainexpresses a mutant form of an A. niger ortholog of the S. cerevisiaeSLN1 gene.181. The method of embodiment 180, wherein a nucleic sequence of themutant form of the A. niger ortholog of the S. cerevisiae SLN1 gene isSEQ ID NO: 13.182. The method of embodiment 179 or 180, wherein the mutant form of theA. niger ortholog of the S. cerevisiae SLN1 gene is operably linked to apromoter sequence selected from SEQ ID NO: 1 or 2.183. The method of any of embodiments 164-182, wherein the fungal strainpossesses a genetic perturbation.184. The method of embodiment 183, wherein the genetic perturbation isselected from single nucleotide polymorphisms, DNA insertions, DNAdeletions or any combination thereof.185. The method of embodiment 183 or 184, wherein the geneticperturbation is introduced into protoplasts derived from the fungalstrain via transforming the protoplasts with a ribonucleoprotein complex(RNP-complex).186. The method of embodiment 185, wherein the RNP-complex comprises anRNA guided endonuclease complexed with a guide RNA (gRNA).187. The method of embodiment 186, wherein the RNA guided endonucleaseis a Class 2 CRISPR-Cas System RNA guided endonuclease.188. The method of embodiment 187, wherein the Class 2 CRISPR-Cas systemRNA guided endonuclease is a Type II, Type V or Type VI RNA guidedendonuclease.189. The method of embodiment 187, wherein the Class 2 CRISPR-Cas systemRNA guided endonuclease is selected from Cas9, Cas12a, Cas12b, Cas12c,Cas12d, Cas12e, Cas13a, Cas13b, Cas13c or homologs, orthologs, mutants,variants or modified versions thereof.190. The method of embodiment 189, wherein the Class 2 CRISPR-Cas systemRNA guided endonuclease is Cas9 or homologs, orthologs or paralogsthereof.191. The method of embodiment 186, wherein the gRNA is a CRISPR RNA(crRNA) alone or annealed to a transactivating CRISPR RNA (tracrRNA).192. The method of embodiment 186, wherein the gRNA is a single guideRNA (sgRNA) comprising a tracrRNA and a crRNA.193. The method of embodiment 191 or 192, wherein the crRNA comprises aguide sequence complementary to a target gene within the genome of thefungal strain, wherein introduction of the RNP-complex into theprotoplasts facilitates introduction of the genetic perturbation intothe target gene.194. The method of embodiment 193, wherein the genetic perturbation ofthe target gene is facilitated by cleavage of the target gene by theRNP-complex to generate DNA ends in the target gene followed bynon-homologous end joining of the DNA ends in the target gene by thenon-homologous end joining (NHEJ) pathway.195. The method of embodiment 193, further comprising co-transforming adonor DNA comprising a mutated version of the target gene, wherein themutated version of the target gene is flanked on both sides bynucleotides homologous to the target gene locus.196. The method of embodiment 195, wherein the genetic perturbation ofthe target gene is facilitated by cleavage of the target gene by theRNP-complex to generate DNA ends in the target gene followed byreplacement of the target gene with the donor DNA via homologousrecombination.197. The method of any of embodiments 185-196, wherein step (b) furthercomprises co-transforming a vector comprising a selectable marker.198. The method of embodiment 197, wherein the selectable marker is usedduring step (d) to select clonal populations derived from transformationcompetent fungal strains.199. The method of embodiment 183 or 184, wherein the geneticperturbation is introduced into protoplasts derived from the fungalstrain by transforming the plurality of protoplasts with a firstconstruct and a second construct, wherein the first construct comprisesa first polynucleotide flanked on both sides by nucleotides homologousto a first locus in the genome of the protoplast and the secondconstruct comprises a second polynucleotide flanked on both sides bynucleotides homologous to a second locus in the genome of theprotoplast, wherein the transformation results in integration of thefirst construct into the first locus and the second construct into thesecond locus by homologous recombination, wherein at least the secondlocus is a first selectable marker gene in the protoplast genome, andwherein the first polynucleotide comprises the genetic perturbation.200. The method of embodiment 199, wherein the selectable marker gene isused during step (d) to facilitate selection of clonal populationsderived from fungal strains comprising the genetic perturbation.201. The method of any of embodiments 195-200, wherein the fungal strainpossesses a non-functional non-homologous end joining (NHEJ) pathway.202. The method of embodiment 201, wherein the NHEJ pathway is madenon-functional by exposing the fungal strain to an antibody, a chemicalinhibitor, a protein inhibitor, a physical inhibitor, a peptideinhibitor, or an anti-sense or RNAi molecule directed against acomponent of the NHEJ pathway.203. The method of embodiment 202, wherein the chemical inhibitor isW-7.204. A method for producing a filamentous fungal strain, the methodcomprising:a.) providing a plurality of protoplasts, wherein the plurality ofprotoplasts were prepared from a culture of a parent filamentous fungalstrain;b.) transforming each protoplast from the plurality of protoplasts witha ribonucleoprotein complex (RNP-complex); andc.) selecting and screening individual filamentous fungal strainsderived from the transformed protoplasts for phenotypic performanceimprovements over the parent filamentous fungal strain, therebyidentifying genetic perturbations in the genome of the selectedindividual filamentous fungal strains that confer phenotypic performanceimprovements.205. The method of embodiment 204, wherein the genetic perturbations areselected from single nucleotide polymorphisms, DNA insertions, DNAdeletions or any combination thereof.206. The method of embodiment 204 or 205, wherein the RNP-complexcomprises an RNA guided endonuclease complexed with a guide RNA (gRNA).207. The method of embodiment 206, wherein the RNA guided endonucleaseis a Class 2 CRISPR-Cas System RNA guided endonuclease.208. The method of embodiment 207, wherein the Class 2 CRISPR-Cas systemRNA guided endonuclease is a Type II, Type V or Type VI RNA guidedendonuclease.209. The method of embodiment 207, wherein the Class 2 CRISPR-Cas systemRNA guided endonuclease is selected from Cas9, Cas12a, Cas12b, Cas12c,Cas12d, Cas12e, Cas13a, Cas13b, Cas13c or homologs, orthologs, mutants,variants or modified versions thereof.210. The method of embodiment 209, wherein the Class 2 CRISPR-Cas systemRNA guided endonuclease is Cas9 or homologs, orthologs or paralogsthereof.211. The method of embodiment 206, wherein the gRNA is a CRISPR RNA(crRNA) alone or annealed to a transactivating CRISPR RNA (tracrRNA).212. The method of embodiment 206, wherein the gRNA is a single guideRNA (sgRNA) comprising a tracrRNA and a crRNA.213. The method of embodiment 211 or 212, wherein the crRNA comprises aguide sequence that is complementary to a target gene within the genomeof the parent filamentous fungal strain, wherein introduction of theRNP-complex perturbs the target gene in the protoplasts.214. The method of embodiment 213, wherein the perturbation of thetarget gene is facilitated by cleavage of the target gene by theRNP-complex to generate DNA ends in the target gene followed bynon-homologous end joining of the DNA ends in the target gene by thenon-homologous end joining (NHEJ) pathway.215. The method of embodiment 213, wherein step (b) further comprisesco-transforming a donor DNA comprising a mutated version of the targetgene, wherein the mutated version of the target gene is flanked on bothsides by nucleotides homologous to the target gene locus.216. The method of embodiment 215, wherein the perturbation of thetarget gene is facilitated by cleavage of the target gene by theRNP-complex to generate DNA ends in the target gene followed byreplacement of the target gene with the donor DNA via homologousrecombination.217. The method of any of embodiments 204-216, wherein step (b) furthercomprises co-transforming a vector comprising a selectable marker.218. The method of embodiment 217, wherein the selectable marker is usedduring step (c) to select transformation competent individualfilamentous fungal strains for subsequent screening for phenotypicperformance improvements over the parent filamentous fungal strain.219. The method of any of one of embodiments 215-218, wherein the parentfilamentous fungal strain possesses a non-functional non-homologous endjoining (NHEJ) pathway.220. The method of embodiment 219, wherein the NHEJ pathway is madenon-functional by exposing the cell to an antibody, a chemicalinhibitor, a protein inhibitor, a physical inhibitor, a peptideinhibitor, or an anti-sense or RNAi molecule directed against acomponent of the NHEJ pathway.221. The method of embodiment 220, wherein the chemical inhibitor isW-7.222. The method of any of embodiments 204-221, wherein the phenotypicperformance improvement of the filamentous fungal strain comprises atleast a 10% increase in a measured phenotypic variable for a product ofinterest compared to the phenotypic performance of the parentfilamentous fungal strain.223. The method of any of embodiments 204-221, wherein the phenotypicperformance improvement of the filamentous fungal strain comprises atleast a one-fold increase in a measured phenotypic variable for aproduct of interest compared to the phenotypic performance of the parentfilamentous fungal strain.224. The method of embodiment 222 or 223, wherein the measuredphenotypic variable is selected from the group consisting of: volumetricproductivity of the product of interest, specific productivity of theproduct of interest, yield of the product of interest, titer of theproduct of interest, and combinations thereof.225. The method of embodiment 222 or 223, wherein the measuredphenotypic variable is increased or more efficient production of theproduct of interest,226. The method of embodiment 222 or 223, wherein the product ofinterest is selected from the group consisting of: a small molecule,enzyme, peptide, amino acid, organic acid, synthetic compound, fuel,alcohol, primary extracellular metabolite, secondary extracellularmetabolite, intracellular component molecule, and combinations thereof.227. The method of any of embodiments 204-226, wherein the parentfilamentous fungal strain is selected from Achlya, Acremonium,Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium,Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus,Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella,Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthorathermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia,Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella speciesor teleomorphs, or anamorphs, and synonyms or taxonomic equivalentsthereof.228. The method of embodiment 227, wherein the filamentous fungal strainis Aspergillus niger or teleomorphs or anamorphs thereof.229. The method of embodiment 228, wherein the filamentous fungal strainpossess a non-mycelium, pellet morphology.230. The method of embodiment 229, wherein the filamentous fungal strainexpresses a mutant form of an A. niger ortholog of the S. cerevisiaeSLN1 gene.231. The method of embodiment 230, wherein a nucleic sequence of themutant form of the A. niger ortholog of the S. cerevisiae SLN1 gene isSEQ ID NO: 13.232. The method of embodiment 230 or 231, wherein the mutant form of theA. niger ortholog of the S. cerevisiae SLN1 gene is operably linked to apromoter sequence selected from SEQ ID NO: 1 or 2.233. The method of any of embodiment 204-232, further comprisinggenerating isolated clonal populations derived from the individualfilamentous fungal strains prior to step (c).234. The method of embodiment 233, wherein the isolating comprises:(i) inducing the transformed protoplasts to produce a plurality offungal spores, wherein each fungal spore form the plurality is derivedfrom a single transformed protoplast;(ii) resuspending the plurality of fungal spores derived from a singletransformed protoplast in a liquid to generate a liquid suspension;(iii) dispensing a discrete volume of the liquid suspension to anindividual reaction area in a substrate comprising a plurality ofreaction areas, wherein each reaction area in the plurality of reactionareas comprises growth media, wherein the dispensing results in aprobability that at least 75% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores; and(iv) culturing the dispensed single viable fungal spores in the reactionareas comprising growth media, thereby generating isolated clonalpopulations derived from the individual filamentous fungal strains.235. The method of embodiments 234, further comprising screening thediscrete volumes for the presence or absence of a single fungal spore inthe discrete volumes, wherein only the discrete volumes containing asingle fungal spore are selected for step (iii).236. The method of embodiment 235, wherein the dispensing results in aprobability that at least 80% of the individual reaction areas containno more than an single viable fungal spore from the plurality of fungalspores.237. The method of embodiment 235, wherein the dispensing results in aprobability that at least 90% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores.238. The method of embodiment 234, wherein the dispensing results in aprobability that at least 95% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores.239. The method of embodiment 234, wherein the dispensing results in aprobability that at least 99% of the individual reaction areas containno more than a single viable fungal spore from the plurality of fungalspores.240. The method of embodiment 234, wherein the dispensing results in aprobability that substantially all of the individual reaction areascontain no more than a single viable fungal spore from the plurality offungal spores.241. The method of embodiment 235, wherein the screening the discretevolumes entails optically distinguishing the presence or absence of asingle fungal spore in the discrete volumes.242. The method of embodiment 241, wherein the screening is performedusing a microfluidic device capable of optically distinguishing thepresence or absence of a single fungal spore in the discrete volumes.243. The method of embodiment 233, wherein the isolating comprises. (i)inducing the transformed protoplasts to produce a plurality of fungalspores, wherein each fungal spore form the plurality is derived from asingle transformed protoplast; (ii) resuspending the plurality of fungalspores derived from a single transformed protoplast in a liquid togenerate a liquid suspension; (iii) diluting the liquid suspension,wherein the dilution is a limiting dilution; (iv) dispensing a discretevolume of the dilution to an individual reaction area in a substratecomprising a plurality of reaction areas, wherein each reaction area inthe plurality of reaction areas comprises growth media, wherein thelimiting dilution results in a probability that the discrete volume ofthe dilution dispensed to each reaction area contains either one or noviable spore follows a Poisson Distribution, whereby greater than 90% ofthe reaction areas in the plurality of reaction areas contain no viablespores and greater than 90% of reaction areas that contain one or moreviable spores contain only a single viable spore; (v) culturing thedispensed single viable fungal spores in the reaction areas comprisinggrowth media; and (vi) selecting clonal populations growing in thereaction areas, thereby isolating clonal populations derived from singlefungal spores.244. The method of any of embodiments 234-243, wherein the reactionareas are present in a microtiter plate.245. The method of embodiment 244, wherein the microtiter plate contains96 wells, 384 wells or 1536 wells.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as an acknowledgment or anyform of suggestion that they constitute valid prior art or form part ofthe common general knowledge in any country in the world.

In addition, the following particular applications are incorporatedherein by reference: U.S. application Ser. No. 15/396,230 (U.S. Pub. No.US 2017/0159045 A1) filed on Dec. 30, 20016; PCT/US2016/065465 (WO2017/100377 A1) filed on Dec. 7, 2016; U.S. application Ser. No.15/140,296 (US 2017/0316353 A1) filed on Apr. 27, 2016;PCT/US2017/029725 (WO 2017/189784 A1) filed on Apr. 26, 2017;PCT/US2016/065464 (WO 2017/100376 A2) filed on Dec. 7, 2016; U.S. Prov.App. No. 62/431,409 filed on Dec. 7, 2016; U.S. Prov. App. No.62/264,232 filed on Dec. 7, 2015; and U.S. Prov. App. No. 62/368,786filed on Jul. 29, 2016. In addition, the following particularapplications are incorporated herein by reference: PCT/US2017/069086,filed on Dec. 29, 2017; and U.S. Prov. App. No. 62/441,040, filed onDec. 30, 2016.

What is claimed is:
 1. A method for isolating clonal populations fromsingle fungal spores, the method comprising: (a) dispensing discretevolumes of a liquid suspension comprising a plurality of fungal sporesfrom a fungal strain to individual reaction areas in a substratecomprising a plurality of reaction areas, wherein each reaction area inthe plurality of reaction areas comprises growth media, wherein thedispensing results in a probability that at least 90% of the individualreaction areas contain either no viable spores or no more than a singleviable fungal spore from the plurality of fungal spores; (b) culturingthe dispensed single viable fungal spores in the reaction areascomprising growth media; and (c) selecting clonal populations growing inthe reaction areas, thereby isolating clonal populations from singlefungal spores.
 2. The method of claim 1, further comprising screeningthe discrete volumes for the presence or absence of a single fungalspore in the discrete volumes, wherein only the discrete volumescontaining a single fungal spore are selected for step (a).
 3. Themethod of claim 2, wherein the dispensing results in a probability thatat least 90%, at least 95%, at least 99% or all of the individualreaction areas contain no more than a single viable fungal spore fromthe plurality of fungal spores.
 4. The method of claim 2, wherein thescreening the discrete volumes comprises optically distinguishing thepresence or absence of a single fungal spore in the discrete volumes. 5.The method of claim 4, wherein the screening is performed using amicrofluidic device capable of optically distinguishing the presence orabsence of a single fungal spore in the discrete volumes.
 6. The methodof claim 1, wherein the liquid suspension comprising a plurality offungal spores from a fungal strain is a limiting dilution, wherein thedispensing of the limiting dilution results in a probability that thediscrete volume of the dilution dispensed to each reaction area containseither one or no viable spore follows a Poisson Distribution, wherebygreater than 90% of the reaction areas in the plurality of reactionareas contain no viable spores and greater than 90% of reaction areasthat contain one or more viable spores contain only a single viablespore.
 7. The method of claim 1, wherein the reaction areas are presentin a microtiter plate, wherein the microtiter plate contains 96 wells,384 wells or 1536 wells.
 8. The method of claim 1, wherein the fungalstrain is a filamentous fungal strain.
 9. The method of claim 8, whereinthe filamentous fungal strain is selected from Achlya, Acremonium,Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium,Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus,Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella,Gliocladium, Humicola, Hypocrea, Myceliophthora (e.g., Myceliophthorathermophila), Mucor, Neurospora, Penicillium, Podospora, Phlebia,Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum,Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia,Tramates, Tolypocladium, Trichoderma, Verticillium, Volvariella species,teleomorphs, anamorphs, and synonyms and taxonomic equivalents thereof.10. The method of claim 9, wherein the filamentous fungal strain isAspergillus niger or teleomorphs or anamorphs thereof.
 11. The method ofclaim 10, wherein the filamentous fungal strain possesses anon-mycelium, pellet morphology.
 12. The method of claim 11, wherein thefilamentous fungal strain expresses a mutant form of an A. nigerortholog of the S. cerevisiae sln1 gene.
 13. The method of claim 12,wherein a nucleic sequence of the mutant form of the A. niger orthologof the S. cerevisiae sln1 gene is SEQ ID NO:
 13. 14. The method of claim11, wherein the mutant form of the A. niger ortholog of the S.cerevisiae sln1 gene is operably linked to a promoter sequence selectedfrom SEQ ID NO: 1 and
 2. 15. The method of claim 1, wherein steps(a)-(c) are automated.
 16. The method of claim 1, wherein steps (a)-(c)are performed using automated robotics.
 17. The method of claim 16,wherein the automated robotics are in communication with one or moreprocessors in a system, wherein the one or more processors are each incommunication with one or more memories.
 18. The method of claim 17,wherein the one or more processors comprise instructions stored thereonthat when executed by the one or more processors cause the system toperform steps (a)-(c).